Novel antibodies

ABSTRACT

The present invention relates to the use of VEGF antagonists and a novel anti-α5β1 antibody for treating cancer and inhibiting angiogenesis and/or vascular permeability, including inhibiting abnormal angiogenesis in diseases. The present invention also relates to compositions and kits comprising novel anti-α5β1 antibodies and methods of making and using them.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication Nos. 60/975,471, filed Sep. 26, 2007, the disclosure ofwhich is hereby incorporated by reference in its entirety for allpurposes.

FIELD OF THE INVENTION

The present invention relates to novel α5β1 antibodies, compositions andkits comprising the antibodies and methods for using the antibodies.

BACKGROUND OF THE INVENTION

α5β1 integrin is a cell membrane glycoprotein that mediates cell-celland cell-ECM interactions through its major ligand, fibronectin. α5β1integrin plays a role in cell migration, differentiation and survivial.Levels of α5β1 integrin are elevated in tumor vascular endothelium(e.g., gastric, colorectal hepatocellular, uterocervial, and breastcarcinomas) and other angiogenic vessels. α5β1 integrin modulates muralcell association with endothelial cells and the assembly of theendothelial extracellular matrix during angiogenesis. As such, α5β1integrin is a useful target for inhibition of angiogenesis andsensitization of cells to the effects of a VEGF antagonist.

Thus, there is a need in the art for compositions and methods fortargeting α5β1 integrin. The present invention meets this and otherneeds.

SUMMARY OF THE INVENTION

The present invention relates to novel anti-α5β1 antibodies derived fromthe antibody produced from the 7H5 hybridoma, kits and compositionscomprising the novel anti-α5β1 antibodies, and methods of making orusing them. The anti-α5β1 antibodies of this invention have improvedbinding to α5β1 compared to the antibody produced from the 7H5hybridoma. Such antibodies include humanized antibodies. According toanother embodiment, the new anti-α5β1 antibodies can be conjugated toanother entity such as, but not limited to, a therapeutic agent or afluorescent dye or other marker to detect α5β1 in patients or in patientsamples. Such new α5β1 antibodies can be used in a variety oftherapeutic and diagnostic methods. For example, such anti-α5β1antibodies can be used in treating abnormal angiogenesis, neoplasia,ocular diseases and autoimmune diseases. Such antibodies can be used fordetecting α5β1 protein in patients or patient samples by contacting suchantibodies to α5β1 protein in patients or in patient samples anddetermining qualitatively or quantitatively the anti-α5β1 antibody boundto the α5β1 protein.

According to one embodiment, the anti-α5β1 antibodies of this inventioncomprise a light chain variable domain sequence comprising (1) an LHVR1comprising the amino acid sequence KASQ-N/S-VGSDVA (SEQ ID NO:10), (2)an LHVR2 comprising the amino acid sequence STSYRYS (SEQ ID NO:11) and(3) an LHVR3 comprising the amino acid sequence QQY-N/S-SYPFT (SEQ IDNO:12). According to another embodiment, the anti-α5β1 antibodies ofthis invention comprise a heavy chain variable domain sequencecomprising (1) an HHVR1 comprising the amino acid sequenceGYTF-T/S-DYYLY (SEQ ID NO:13), (2) an HHVR2 comprising the amino acidsequence GISPS-N/S-GGTTF-N/A-D-N/A-FE-N/G (SEQ ID NO:14) and (3) anHHVR3 comprising the amino acid sequence DAYGDWYFDV (SEQ ID NO:15).

According to another embodiment, the anti-α5β1 antibodies of thisinvention comprise a light chain variable domain having the sequence SEQID NO:1 or variant there of and a heavy chain variable domain having thesequence SEQ ID NO:6 or variant thereof. According to one embodiment thevariant sequence of the heavy chain variable domain comprises an aminoacid substitution at a residue selected from the group consisting of 30,48, 49, 54, 60, 62, 65, 66, 67 and 69 (Kabat numbering system).According to another embodiment the variant sequence of the light chainvariable domain comprises an amino acid substitution at a residueselected from the group consisting of 28, 46 and 92 (Kabat numberingsystem). According to one embodiment, the heavy chain variable domaincomprises an amino acid substitution selected from the group consistingof T30S, I48V, G49S, N54S, N60A, N62A, N62S, N65G, K66R, A67F and L69I(Kabat numbering system). According to one embodiment, the light chainvariable domain comprises an amino acid substitution selected from thegroup consisting of N28S, T46L and N92S (Kabat numbering system).

The present invention also relates to a composition comprising apolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, and 9. According toanother embodiment, the composition comprises an antibody comprising thelight chain variable domain sequence SEQ ID NO:1 and the heavy chainvariable domain sequence SEQ ID NO:5. According to another embodiment,the composition comprises an antibody comprising the light chainvariable domain sequence SEQ ID NO:2 and the heavy chain variable domainsequence SEQ ID NO:6. According to another embodiment, the compositioncomprises an antibody comprising the light chain variable domainsequence SEQ ID NO:2 and the heavy chain variable domain sequence SEQ IDNO:7. According to another embodiment, the composition comprises anantibody comprising the light chain variable domain sequence SEQ ID NO:3and the heavy chain variable domain sequence SEQ ID NO:8. According toanother embodiment, the composition comprises an antibody comprising thelight chain variable domain sequence SEQ ID NO:4 and the heavy chainvariable domain sequence SEQ ID NO:9.

According to yet another embodiment, the present invention provides amethod of treating cancer in a subject comprising the step(s) ofadministering a VEGF antagonist and an anti-α5β1 antibody of thisinvention. According to one preferred embodiment, the cancer isresponsive to VEGF antagonist therapies. In another embodiment, a methodof treating age related macular degeneration (AMD), including wetage-related macular degeneration, in a subject suffering from AMDcomprising the step(s) of administering a therapeutically effectiveamount of a VEGF antagonist and the anti-α5β1 antibody of thisinvention. In yet another embodiment, a method of treating an autoimmunedisease in a subject comprising the step(s) of administering atherapeutically effective amount of a VEGF antagonist and the anti-α5β1antibody.

In one embodiment, the subject to be treated may be administered theVEGF antagonist initially and subsequently treated with the anti-α5β1antibody of this invention. In another embodiment, the subject istreated with the VEGF antagonist and the anti-α5β1 antibody of thisinvention within the cycles of treatment with either drug. According toanother embodiment, the subject is treated with the VEGF antagonistuntil the subject is unresponsive to VEGF antagonist treatment and thenthe subject is treated with the anti-α5β1 antibody of this invention. Inone particular embodiment, the subject is treated with the VEGFantagonist when the cancer is non-invasive or early stage and treatedwith the anti-α5β1 antibody of this invention when the cancer isinvasive. In another embodiment, subject being treated with theanti-α5β1 antibody of this invention has elevated α5β1 levels in adiseased tissue compared to tissue from a subject not suffering from thedisease. In this instance, the method can further include the step ofdetecting α5β1 in the subject, e.g., in a diseased tissue aftertreatment with a VEGF antagonist. According to one embodiment, theinvasive cancer is a metastasized cancer. According to anotherembodiment, the early stage cancer is a cancer treated by adjuvanttherapy (e.g., chemotherapy or surgical removal).

In one preferred embodiment, the subject is suffering from a diseasehaving abnormal angiogenesis. According to another embodiment, thedisease is selected from the group consisting of a cancer, an immunedisease or an ocular disease. According to one preferred embodiment, thedisease is selected from the group consisting of a solid tumor, ametastatic tumor, a soft tissue tumor, a disease having ocularneovascularisation, an inflammatory disease having abnormalangiogenesis, a disease arising after transplantation into the subjectand a disease having abnormal proliferation of fibrovascular tissue.According to another preferred embodiment, the cancer is selected fromthe group consisting of breast cancer (including metastatic breastcancer), cervical cancer, colorectal cancer (including metastaticcolorectal cancer), lung cancer (including non-small cell lung cancer),non-Hodgkins lymphoma (NHL), chronic lymphocytic leukemia, renal cellcancer, prostate cancer including hormone refractory prostate cancer,liver cancer, head and neck cancer, melanoma, ovarian cancer,mesothelioma, soft tissue cancer, gastrointestinal stromal tumor,glioblastoma multiforme and multiple myeloma. According to anotherpreferred embodiment, the disease is selected from the group consistingof retinopathy, age-related macular degeneration (e.g., wet AMD),diabetic macular edema, retinal vein occlusion (RVO), and dryAMD/geographic atrophy (for prevention of progression to wet AMD),rubeosis; psoriasis, an inflammatory renal disease, haemolytic uremicsyndrome, diabetic nephropathy (e.g., proliferative diabeticretinopathy), arthritis (e.g., psoriatic arthritis, osteoarthritis,rheumatoid arthritis), inflammatory bowel disease, chronic inflammation,chronic retinal detachment, chronic uveitis, chronic vitritis, cornealgraft rejection, corneal neovascularization, corneal graftneovascularization, Crohn's disease, myopia, ocular neovascular disease,Pagets disease, pemphigoid, polyarteritis, post-laser radial keratotomy,retinal neovascularization, Sogrens syndrome, ulcerative colitis, graftrejection, lung inflammation, nephrotic syndrome, edema, ascitesassociated with malignancies, stroke, angiofibroma and neovascularglaucoma. In one embodiment, the subject is further administered atherapeutic agent selected from the group consisting of ananti-neoplastic agent, a chemotherapeutic agent and a cytotoxic agent.

According to one preferred embodiment of this invention, the subject tobe treated with the anti-α5β1 antibody is suffering from a relapse afterVEGF antagonist treatment or has become refractory to VEGF antagonisttreatment. According to another embodiment, the subject to be treatedwith the anti-α5β1 antibody of this invention and a VEGF antagonist issuffering from a metastatic cancer or has previously been treated withadjuvant therapy. In one embodiment, the candidate patient is relapsed,refractory or resistant to a chemotherapeutic agent such as irinotecan.Examples of such diseases, include but are not limited to, metastaticcolorectal cancer, relapsed metastatic colorectal cancer, metastaticbreast cancer, relapsed metastatic breast cancer, metastatic HER2+breast cancer, adjuvant breast cancer, adjuvant HER2+ breast cancer,metastatic pancreatic cancer, adjuvant colon cancer, adjuvant non-smallcell lung cancer, adjuvant rectal cancer, adjuvant non small cell lungcancer, metastatic non small cell lung cancer, metastatic ovariancancer, metastatic renal cell cancer and adjuvant renal cell cancer.

According to one embodiment, the subject suffering from a diseasedescribed herein is administered a maintenance therapy after treatmentfor the disease with a VEGF antagonist, wherein the maintenance therapyis the anti-α5β1 antibody of this invention alone or sequentially orconcurrently with a VEGF antagonist.

According to one preferred embodiment, the VEGF antagonist can beselected from the group consisting of an antibody, an immunoadhesin, apeptibody, a small molecule and a nucleic acid that hybridizes to anucleic acid molecule encoding VEGF under stringent conditions (e.g.,ribozyme, siRNA and aptamer). According to one preferred embodiment, theVEGF antagonist is an antibody. According to another embodiment, theantibody is a monoclonal antibody. According to one preferredembodiment, the anti-VEGF antibody is capable of being competitivelyinhibited from binding to human VEGF by the Avastin® antibody. Accordingto another embodiment, the anti-VEGF antibody is human, humanized orchimeric. According one specific embodiment, the anti-VEGF antibody isthe Avastin® antibody. According to another embodiment, the anti-VEGFantibody is selected from the group consisting of a Fab, Fab′, aF(ab)′₂, single-chain Fv (scFv), an Fv fragment; a diabody and a linearantibody. According to another embodiment, the VEGF antagonist is abispecific antibody that binds VEGF and comprises a heavy chain andlight chain variable domain of the anti-α5β1 antibody of this invention.

According to one preferred embodiment, the anti-α5β1 antibody of thisinvention is an antibody comprising an Fc portion of a human IgG.According to another embodiment, the anti-α5β1 antibody of thisinvention comprises the CH1, CH2 and CH3 domain of a human IgG1 orhIgG4. According to one preferred embodiment, the anti-α5β1 antibody ishumanized antibody. According one specific embodiment, the anti-α5β1antibody is the 7H5 antibody or a chimeric or humanized antibodythereof. According to another embodiment, the anti-α5β1 antibody isselected from the group consisting of a Fab, Fab′, a F(ab)′₂,single-chain Fv (scFv), an Fv fragment; a diabody and a linear antibody.According to another embodiment, the anti-α5β1 antibody of thisinvention is a bispecific antibody that binds VEGF and α5β1 and is aVEGF antagonist. According to yet another embodiment, the anti-α5β1antibody of this invention has an altered effector function. Accordingone embodiment, an anti-α5β1 antibody is altered to decrease or preventantibody dependent cellular cytotoxicity (ADCC) or complement dependentcytotoxicity (CDC) activity (e.g., by altering the nucleic acid sequenceencoding the Fc portion of the antibody). According to yet anotherembodiment, the anti-α5β1 antibody has been altered to increase ordecrease its half-life in humans (e.g., by altering the nucleic acidsequence encoding the Fc portion of the antibody).

According to one embodiment, the α5β1 antibody is conjugated to acytotoxic agent or a chemotherapeutic agent. According to anotherembodiment, the cytotoxic agent is a radioactive isotope or a toxin.

The present invention provides compositions comprising a VEGFantagonist, the α5β1 antibody of this invention and a pharmaceuticallyacceptable carrier. The present invention also provides articles ofmanufacture comprising instructions for detecting α5β1 in a subject whohas been treated with a VEGF antagonist.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts alignment of sequences of the light-chain variable domainfor the following with respect to the anti-integrin α5β1 clone 7H5:humanized 7H5 antibody based on grafting CDRs of murine 7H5 (h7H5.v1)(SEQ ID NO:1), humanized 7H5 antibody based on the replacement ofresidues in CDR-L1, -L3 and -H2 of h7H5.v1 (h7H5.v2, SEQ ID NO:2) andhumanized 7H5 antibodies based on framework modification on h7H5.v2(h7H5.v4 (SEQ ID NO:3) and h7H5.v5 (SEQ ID NO:4)). The h7H5.v3 lightchain variable domain is identical in sequence to SEQ ID NO:2.

FIG. 2 depicts alignment of sequences of the heavy-chain variable domainfor the following with respect to the anti-integrin α5β1 clone 7H5:humanized 7H5 antibody based on grafting CDRs of murine 7H5 (h7H5.v1)(SEQ ID NO:5), humanized 7H5 antibody based on the replacement ofresidues in CDR-L1, -L3 and -H2 of h7H5.v1 (h7H5.v2) (SEQ ID NO:6) andhumanized 7H5 antibodies based on framework modification on h7H5.v2(h7H5.v4 (SEQ ID NO:8) and h7H5.v5 (SEQ ID NO:9)). The h7H5.v3 heavychain variable domain is identical in sequence to h7H5.v2, except thatit has an N62S amino acid mutation (h7H5.v3 VH sequence designated SEQID NO:7).

FIG. 3 depicts the results of a BIACORE® analysis of chimeric 7H5-IgGand humanized 7H5.v1-IgG against human integrin α5β1. (A) Chimeric7H5-IgG and humanized 7H5.v1-IgG were immobilized on two different flowcells of CM5 sensor chip of 450RU (Response Unit), and 2-fold serialdiluted human integrin α5β1 from 300 nM to 0.29 nM were injected throughsensor chip to determine binding affinities and kinetics at 25° C. (B)Human integrin α5β1 were immobilized on the CM5 sensor chip of 800RU,and 2-fold serial diluted chimeric 7H5-IgG and humanized 7H5.v1-IgG from200 nM to 0.2 nM were injected through sensor chip to determine bindingaffinities and kinetics at 25° C.

FIG. 4 depicts a phage competition ELISA to determine phage IC50 againsthuman integrin α5β1 for humanized 7H5.v1 single point mutant clonedisplayed on the phage as a monovalent Fab format.

FIG. 5 depicts the summary of relative fold of changes of bindingaffinity (IC50) for each single point mutant to the parental cloneh7H5.v1.

FIG. 6 depicts a phage competition ELISA to determine phage IC50 againsthuman integrin α5 μl for humanized 7H5.v1 multiple substitution clonesdisplayed on the phage as a monovalent Fab format. The first twovariants were generated based on the selection from FIG. 5 ashighlighted. Overall, all six variants retained and showed slightlyimproved binding affinity; therefore, two variants, h7H5.v2 and h7H5.v3as indicated were selected to include glycine substitution at position65 of CDR-H2.

FIG. 7 depicts the results of a BIACORE® analysis of humanized 7H5.v1,v2 and v3-IgG against human integrin α5β1. Humanized 7H5.v1, v2 andv3-IgG were immobilized on three different flow cells of CM5 sensor chipof 450RU (Response Unit), and 2-fold serial diluted human integrin α5 μlfrom 300 nM to 0.29 nM were injected through sensor chip to determinebinding affinities and kinetics at 25° C. Humanized 7H5.v2 indicates themost binding affinity improvement in off-rate.

FIG. 8 depicts a phage competition ELISA to determine phage IC50 againsthuman integrin α5 μl for humanized 7H5.v2 framework modification clonesdisplayed on the phage as a monovalent Fab format. Most of the frameworksubstitutions showed comparable binding affinity as compared to h7H5.v2,except for position 49 and 78 of heavy chain with changing glycine toserine and alanine to leucine respectively.

FIG. 9 depicts the results of a BIACORE® analysis of humanized 7H5.v2,v4 and v5-IgG against human integrin α5β1. (A) Humanized 7H5.v2, v4 andv5-IgG were immobilized on three different flow cells of CM5 sensor chipof 450RU (Response Unit), and 2-fold serial diluted human integrin α5β1from 300 nM to 0.29 nM were injected through sensor chip to determinebinding affinities and kinetics at 25° C. (B) Human integrin α5β1 wereimmobilized on the CM5 sensor chip of 800RU, and 2-fold serial dilutedhumanized 7H5.v2, v4 and v5-IgG from 200 nM to 0.2 nM were injectedthrough sensor chip to determine binding affinities and kinetics at 25°C. In both formats, humanized 7H5.v4 shows comparable binding affinityas compared to humanized 7H5.v2.

FIG. 10 depicts the results of skin wound healing experimentsdemonstrating that administration of the combination of h7H5.v2 andanti-VEGF enhances the anti-angiogenic effects of anti-VEGF alone.

FIG. 11 depicts the results of skin wound healing experimentsdemonstrating that administration of the combination of h7H5.v4 andanti-VEGF enhances the anti-angiogenic effects of anti-VEGF alone.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

The present invention is based on the identification of novel antibodiesthat bind α5β1 integrin. The α5β1 antibodies are derived from themonoclonal antibody 7H5 and can be used in a variety of therapeutic anddiagnostic methods. For example, the α₅β₁ antibodies can be used aloneor in combination with other agents in treating abnormal angiogenesis,neoplasia, ocular diseases and autoimmune diseases. The antibodies canalso be used for detecting α5β1 protein in patients or patient samplesby administering the antibodies to α₅β₁ protein in patients anddetecting the anti-α₅β₁ antibody bound to the α₅β₁ protein in a samplefrom the patient (e.g., in vivo or ex vivo) or by contacting theantibodies with samples from patients and detecting qualitatively orquantitatively the anti-α₅β₁ antibody bound to the α₅β₁ protein.

II. Definitions

“Alpha5beta1” or “α5β1” or “a5b1” or “α₅β₁” is an integrin comprisingtwo different proteins (i.e., subunits Alpha5 and beta1). α5β1 has beenshown to bind to fibronectin, L1-CAM and fibrinogen. α5β1 integrin isalso known as Very Late Activation-5, VLA-5, alpha5beta1, CD49e/CD29,fibronectin receptor, FNR and GPIc-IIa. According to a preferredembodiment, the α5 μl is a human α5β1.

“Alpha5” is used herein interchangeably with CD49e, α5, integrin alpha5subunit, VLA-5 alpha subunit, IC subunit of GPIc-IIa and FNR alpha chainrefers to one subunit of the α₅β₁ integrin. Alpha5 has four isoformsgenerated by alternative splicing (A-D) and which vary within theircytoplasmic domains. Amino acid sequences for human isoforms of alpha5can be found at, e.g., Genbank accession numbers: X07979, U33879, U33882and U33880, respectively.

“Beta1” also called CD29, beta1, Platelet GPIIa; VLA-beta chain; beta-1integrin chain, CD29; FNRB; MDF2; VLAB; GPIIA; MSK12 and VLA5B. Aminoacid sequences for human Beta1 can be found, e.g., at Genbank AccessionNo. X06256.

The term “VEGF” as used herein refers to the 165-amino acid humanvascular endothelial cell growth factor and related 121-, 189-, and206-amino acid human vascular endothelial cell growth factors, asdescribed by Leung et al. Science, 246:1306 (1989), and Houck et al.Mol. Endocrin., 5:1806 (1991), together with the naturally occurringallelic and processed forms thereof. The term “VEGF” also refers toVEGFs from non-human species such as mouse, rat or primate. Sometimesthe VEGF from a specific species are indicated by terms such as hVEGFfor human VEGF, mVEGF for murine VEGF, and etc. The term “VEGF” is alsoused to refer to truncated forms of the polypeptide comprising aminoacids 8 to 109 or 1 to 109 of the 165-amino acid human vascularendothelial cell growth factor. Reference to any such forms of VEGF maybe identified in the present application, e.g., by “VEGF (8-109),” “VEGF(1-109)” or “VEGF₁₆₅.” The amino acid positions for a “truncated” nativeVEGF are numbered as indicated in the native VEGF sequence. For example,amino acid position 17 (methionine) in truncated native VEGF is alsoposition 17 (methionine) in native VEGF. The truncated native VEGF hasbinding affinity for the KDR and Flt-1 receptors comparable to nativeVEGF. According to a preferred embodiment, the VEGF is a human VEGF.

A “VEGF antagonist” refers to a molecule capable of neutralizing,blocking, inhibiting, abrogating, reducing or interfering with VEGFactivities including its binding to VEGF or one or more VEGF receptorsor the nucleic acid encoding them. Preferrably, the VEGF antagonistbinds VEGF or a VEGF receptor. VEGF antagonists include anti-VEGFantibodies and antigen-binding fragments thereof, polypeptides that bindVEGF and VEGF receptors and block ligand-receptor interaction (e.g.,immunoadhesins, peptibodies), anti-VEGF receptor antibodies and VEGFreceptor antagonists such as small molecule inhibitors of the VEGFRtyrosine kinases, aptamers that bind VEGF and nucleic acids thathybridize under stringent conditions to nucleic acid sequences thatencode VEGF or VEGF receptor (e.g., RNAi). According to one preferredembodiment, the VEGF antagonist binds to VEGF and inhibits VEGF-inducedendothelial cell proliferation in vitro. According to one preferredembodiment, the VEGF antagonist binds to VEGF or a VEGF receptor withgreater affinity than a non-VEGF or non-VEGF receptor. According to onepreferred embodiment, the VEG antagonist binds to VEGF or a VEGFreceptor with a Kd of between 1 uM and 1 pM. According to anotherpreferred embodiment, the VEGF antagonist binds to VEGF or a VEGFreceptor between 500 nM and 1 pM.

According a preferred embodiment, the VEGF antagonist is selected fromthe group consisting of a polypeptide such as an antibody, a peptibody,an immunoadhesin, a small molecule or an aptamer. In a preferredembodiment, the antibody is an anti-VEGF antibody such as the AVASTIN®antibody or an anti-VEGF receptor antibody such as an anti-VEGFR2 or ananti-VEGFR3 antibody. Other examples of VEGF antagonists include:VEGF-Trap, Mucagen, PTK787, SU11248, AG-013736, Bay 439006 (sorafenib),ZD-6474, CP632, CP-547632, AZD-2171, CDP-171, SU-14813, CHIR-258,AEE-788, SB786034, BAY579352, CDP-791, EG-3306, GW-786034,RWJ-417975/CT6758 and KRN-633.

An “anti-VEGF antibody” is an antibody that binds to VEGF withsufficient affinity and specificity. Preferably, the anti-VEGF antibodyof the invention can be used as a therapeutic agent in targeting andinterfering with diseases or conditions wherein the VEGF activity isinvolved. An anti-VEGF antibody will usually not bind to other VEGFhomologues such as VEGF-B or VEGF-C, nor other growth factors such asP1GF, PDGF or bFGF. A preferred anti-VEGF antibody is a monoclonalantibody that binds to the same epitope as the monoclonal anti-VEGFantibody A4.6.1 produced by hybridoma ATCC HB 10709. More preferably theanti-VEGF antibody is a recombinant humanized anti-VEGF monoclonalantibody generated according to Presta et al. (1997) Cancer Res.57:4593-4599, including but not limited to the antibody known asbevacizumab (BV; Avastin®). According to another embodiment, anti-VEGFantibodies that can be used include, but are not limited to theantibodies disclosed in WO 2005/012359. According to one embodiment, theanti-VEGF antibody comprises the variable heavy and variable lightregion of any one of the antibodies disclosed in FIGS. 24, 25, 26, 27and 29 of WO 2005/012359 (e.g., G6, G6-23, G6-31, G6-23.1, G6-23.2, B20,B20-4 and B20.4.1). In another preferred embodiment, the anti-VEGFantibody known as ranibizumab is the VEGF antagonist administered forocular disease such as diabetic retinopathy and wet AMD.

The anti-VEGF antibody “Bevacizumab (BV)”, also known as “rhuMAb VEGF”or “Avastin®”, is a recombinant humanized anti-VEGF monoclonal antibodygenerated according to Presta et al. (1997) Cancer Res. 57:4593-4599. Itcomprises mutated human IgG1 framework regions and antigen-bindingcomplementarity-determining regions from the murine anti-hVEGFmonoclonal antibody A.4.6.1 that blocks binding of human VEGF to itsreceptors. Approximately 93% of the amino acid sequence of Bevacizumab,including most of the framework regions, is derived from human IgG1, andabout 7% of the sequence is derived from the murine antibody A4.6.1.Bevacizumab has a molecular mass of about 149,000 daltons and isglycosylated. Other anti-VEGF antibodies include the antibodiesdescribed in U.S. Pat. No. 6,884,879 and WO 2005/044853.

The anti-VEGF antibody Ranibizumab or the LUCENTIS® antibody or rhuFabV2 is a humanized, affinity-matured anti-human VEGF Fab fragment.Ranibizumab is produced by standard recombinant technology methods in E.coli expression vector and bacterial fermentation. Ranibizumab is notglycosylated and has a molecular mass of ˜48,000 daltons. See WO98/45331and U.S. 2003/0190317.

The alpha5/beta1 monoclonal antibody known as 7H5 was deposited in theATCC as 7H5.4.2.8 (ATCC No. PTA-7421) on Mar. 7, 2006.

Molecules, such as antibodies, characterized by binding to overlappingor the similar areas on a target can be identified by competitiveinhibition/binding assays.

In one embodiment, HUVEC or other cells expressing α₅β₁ are used in acompetitive inhibition assay and FACS is used to evaluate bindinglocalities of two anti-α₅β₁ antibodies relative to each other. Forexample, HUVEC cells can be washed in conical tube and spun 5 min @ 1000rpm. The pellet is typically washed two times. Then, the cells can beresuspended, counted and kept on ice until use. 100 ul of a firstanti-α₅β₁ antibody (e.g., start at a 1 ug/ml concentration or lowerconcentration) can be added to the well. Next, 100 μl (e.g., 20×10⁵cells) of cells can be added into per well and incubated on ice for 30min. Next, 100 μl of a biotinylated anti-α₅β₁ antibody (5 μg/ml stock)can be added to each well and incubated on ice for 30 min. The cells arethen washed and pelleted for 5 min. @ 1000 rpm. The supernatant isaspirated. A secondary reagent, R-Phycoerythrin conjugated streptavidin(Jackson 016-110-084), is added to the well (100 μA @ 1:1000). Next, theplate can be wrapped in foil and incubated on ice 30 min. Following theincubation, the pellet can be washed and pelleted 5 min. @ 1000 rpm. Thepellet can be resuspended and transferred to micro titertubes for FACSanalysis.

An “angiogenic factor or agent” is a growth factor which stimulates thedevelopment of blood vessels, e.g., promote angiogenesis, endothelialcell growth, stability of blood vessels, and/or vasculogenesis, etc. Forexample, angiogenic factors, include, but are not limited to, e.g., VEGFand members of the VEGF family, P1GF, PDGF family, fibroblast growthfactor family (FGFs), TIE ligands (Angiopoietins), ephrins, Del-1,fibroblast growth factors: acidic (aFGF) and basic (bFGF), Follistatin,Granulocyte colony-stimulating factor (G-CSF), Hepatocyte growth factor(HGF)/scatter factor (SF), Interleukin-8 (IL-8), Leptin, Midkine,Placental growth factor, Platelet-derived endothelial cell growth factor(PD-ECGF), Platelet-derived growth factor, especially PDGF-BB orPDGFR-beta, Pleiotrophin (PTN), Progranulin, Proliferin, Transforminggrowth factor-alpha (TGF-alpha), Transforming growth factor-beta(TGF-beta), Tumor necrosis factor-alpha (TNF-alpha), Vascularendothelial growth factor (VEGF)/vascular permeability factor (VPF),etc. Angiogenic factors also include factors that accelerate woundhealing, such as growth hormone, insulin-like growth factor-I (IGF-I),VIGF, epidermal growth factor (EGF), CTGF and members of its family, andTGF-alpha and TGF-beta. See, e.g., Klagsbrun and D'Amore, Annu. Rev.Physiol., 53:217-39 (1991); Streit and Detmar, Oncogene, 22:3172-3179(2003); Ferrara & Alitalo, Nature Medicine 5(12):1359-1364 (1999);Tonini et al., Oncogene, 22:6549-6556 (2003) (e.g., Table 1 listingknown angiogenic factors); and, Sato Int. J. Clin. Oncol., 8:200-206(2003).

The “Kd” or “Kd value” for an anti-VEGF antibody according to thisinvention is in one preferred embodiment measured by a radiolabeled VEGFbinding assay (RIA) performed with the Fab version of the antibody and aVEGF molecule as described by the following assay that measures solutionbinding affinity of Fabs for VEGF by equilibrating Fab with a minimalconcentration of (¹²⁵I)-labeled VEGF(109) in the presence of a titrationseries of unlabeled VEGF, then capturing bound VEGF with an anti-Fabantibody-coated plate (Chen, et al., (1999) J. Mol. Biol 293:865-881).To establish conditions for the assay, microtiter plates (Dynex) arecoated overnight with 5 ug/ml of a capturing anti-Fab antibody (CappelLabs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked with2% (w/v) bovine serum albumin in PBS for two to five hours at roomtemperature (approximately 23° C.). In a non-adsorbant plate (Nunc#269620), 100 μM or 26 μM [¹²⁵I]VEGF(109) are mixed with serialdilutions of a Fab of interest, e.g., Fab-12 (Presta et al., (1997)Cancer Res. 57:4593-4599). The Fab of interest is then incubatedovernight; however, the incubation may continue for 65 hours to insurethat equilibrium is reached. Thereafter, the mixtures are transferred tothe capture plate for incubation at room temperature for one hour. Thesolution is then removed and the plate washed eight times with 0.1%Tween-20 in PBS. When the plates had dried, 150 μl/well of scintillant(MicroScint-20; Packard) is added, and the plates are counted on aTopcount gamma counter (Packard) for ten minutes. Concentrations of eachFab that give less than or equal to 20% of maximal binding are chosenfor use in competitive binding assays. According to another embodimentthe Kd or Kd value is measured by using surface plasmon resonance assaysusing a BIAcore™-2000 or a BIAcore™-3000 (BIAcore, Inc., Piscataway,N.J.) at 25° C. with immobilized hVEGF (8-109) CM5 chips at ˜10 responseunits (RU). Briefly, carboxymethylated dextran biosensor chips (CM5,BIAcore Inc.) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Human VEGF is diluted with 10 mM sodium acetate, pH 4.8, into 5 ug/ml(˜0.2 μM) before injection at a flow rate of 5 μl/minute to achieveapproximately 10 response units (RU) of coupled protein. Following theinjection of human VEGF, 1M ethanolamine is injected to block unreactedgroups. For kinetics measurements, two-fold serial dilutions of Fab(0.78 nM to 500 nM) are injected in PBS with 0.05% Tween 20 (PBST) at25° C. at a flow rate of approximately 25 μl/min. Association rates(k_(on)) and dissociation rates (k_(off)) are calculated using a simpleone-to-one Langmuir binding model (BIAcore Evaluation Software version3.2) by simultaneous fitting the association and dissociationsensorgram. The equilibrium dissociation constant (Kd) was calculated asthe ratio k_(off)/k_(on). See, e.g., Chen, Y., et al., (1999) J. Mol.Biol 293:865-881. If the on-rate exceeds 10⁶ M⁻¹ S⁻¹ by the surfaceplasmon resonance assay above, then the on-rate is can be determined byusing a fluorescent quenching technique that measures the increase ordecrease in fluorescence emission intensity (excitation=295 nm;emission=340 nm, 16 nm band-pass) at 25° C. of a 20 nM anti-VEGFantibody (Fab form) in PBS, pH 7.2, in the presence of increasingconcentrations of human VEGF short form (8-109) or mouse VEGF asmeasured in a spectrometer, such as a stop-flow equipped spectrophometer(Aviv Instruments) or a 8000-series SLM-Aminco spectrophotometer(ThermoSpectronic) with a stirred cuvette. Similar binding assays can beperformed for determining the Kd of an anti-α5β1 Fab or antibody usingα5β1 as the target.

As used herein, a subject to be treated is a mammal (e.g., human,non-human primate, rat, mouse, cow, horse, pig, sheep, goat, dog, cat,etc.). The subject may be a clinical patient, a clinical trialvolunteer, an experimental animal, etc. The subject may be suspected ofhaving or at risk for having a cancer, an immune disease, or any otherdisease having abnormal angiogenesis, be diagnosed with a cancer, immunedisease, or any other disease having abnormal angiogenesis. Manydiagnostic methods for cancer, immune disease or any other diseaseexhibiting abnormal angiogenesis and the clinical delineation of thosediseases are known in the art. According to one preferred embodiment,the subject to be treated according to this invention is a human.

The term “abnormal angiogenesis” occurs when new blood vessels groweither excessively or otherwise inappropriately (e.g., the location,timing, degree, or onset of the angiogenesis being undesired from amedical standpoint) in a diseased state or such that it causes adiseased state. In some cases, excessive, uncontrolled, or otherwiseinappropriate angiogenesis occurs when there is new blood vessel growththat contributes to the worsening of the diseased state or cause of adiseased state, such as in cancer, especially vascularized solid tumorsand metastatic tumors (including colon, lung cancer (especiallysmall-cell lung cancer), or prostate cancer), diseases caused by ocularneovascularisation, especially diabetic blindness, retinopathies,primarily diabetic retinopathy or age-induced macular degeneration,choroidal neovascularization (CNV), diabetic macular edema, pathologicalmyopia, von Hippel-Lindau disease, histoplasmosis of the eye, CentralRetinal Vein Occlusion (CRVO), corneal neovascularization, retinalneovascularization and rubeosis; psoriasis, psoriatic arthritis,haemangioblastoma such as haemangioma; inflammatory renal diseases, suchas glomerulonephritis, especially mesangioproliferativeglomerulonephritis, haemolytic uremic syndrome, diabetic nephropathy orhypertensive nephrosclerosis; various imflammatory diseases, such asarthritis, especially rheumatoid arthritis, inflammatory bowel disease,psorsasis, sarcoidosis, arterial arteriosclerosis and diseases occurringafter transplants, endometriosis or chronic asthma and more than 70other conditions. The new blood vessels can feed the diseased tissues,destroy normal tissues, and in the case of cancer, the new vessels canallow tumor cells to escape into the circulation and lodge in otherorgans (tumor metastases). The present invention contemplates treatingthose patients that are at risk of developing the above-mentionedillnesses.

“Abnormal vascular permeability” occurs when the flow of fluids,molecules (e.g., ions and nutrients) and cells (e.g., lymphocytes)between the vascular and extravascular compartments is excessive orotherwise inappropriate (e.g., the location, timing, degree, or onset ofthe vascular permeability being undesired from a medical standpoint) ina diseased state or such that it causes a diseased state. Abnormalvascular permeability may lead to excessive or otherwise inappropriate“leakage” of ions, water, nutrients, or cells through the vasculature.In some cases, excessive, uncontrolled, or otherwise inappropriatevascular permeability or vascular leakage exacerbates or induces diseasestates including, e.g., edema associated with tumors including, e.g.,brain tumors; ascites associated with malignancies; Meigs' syndrome;lung inflammation; nephrotic syndrome; pericardial effusion; pleuraleffusion; permeability associated with cardiovascular diseases such asthe condition following myocardial infarctions and strokes and the like.The present invention contemplates treating those patients that havedeveloped or are at risk of developing the diseases and disordersassociated with abnormal vascular permeability or leakage.

Other patients that are candidates for receiving the antibodies orpolypeptides of this invention have, or are at risk for developing,abnormal proliferation of fibrovascular tissue, acne rosacea, acquiredimmune deficiency syndrome, artery occlusion, atopic keratitis,bacterial ulcers, Bechets disease, blood borne tumors, carotidobstructive disease, choroidal neovascularization, chronic inflammation,chronic retinal detachment, chronic uveitis, chronic vitritis, contactlens overwear, corneal graft rejection, corneal neovascularization,corneal graft neovascularization, Crohn's disease, Eales disease,epidemic keratoconjunctivitis, fungal ulcers, Herpes simplex infections,Herpes zoster infections, hyperviscosity syndromes, Kaposi's sarcoma,leukemia, lipid degeneration, Lyme's disease, marginal keratolysis,Mooren ulcer, Mycobacteria infections other than leprosy, myopia, ocularneovascular disease, optic pits, Osler-Weber syndrome(Osler-Weber-Rendu, osteoarthritis, Pagets disease, pars planitis,pemphigoid, phylectenulosis, polyarteritis, post-laser complications,protozoan infections, pscudoxanthoma elasticum, pterygium keratitissicca, radial keratotomy, retinal neovascularization, retinopathy ofprematurity, retrolental fibroplasias, sarcoid, scleritis, sickle cellanemia, Sogrens syndrome, solid tumors, Stargarts disease, Steven'sJohnson disease, superior limbic keratitis, syphilis, systemic lupus,Terrien's marginal degeneration, toxoplasmosis, trauma, tumors of Ewingsarcoma, tumors of neuroblastoma, tumors of osteosarcoma, tumors ofretinoblastoma, tumors of rhabdomyosarcoma, ulcerative colitis, veinocclusion, Vitamin A deficiency and Wegeners sarcoidosis, undesiredangiogenesis associated with diabetes, parasitic diseases, abnormalwound healing, hypertrophy following surgery, injury or trauma,inhibition of hair growth, inhibition of ovulation and corpus luteumformation, inhibition of implantation and inhibition of embryodevelopment in the uterus.

Anti-angiogenesis therapies are useful in the general treatment of graftrejection, lung inflammation, nephrotic syndrome, preeclampsia,pericardial effusion, such as that associated with pericarditis, andpleural effusion, diseases and disorders characterized by undesirablevascular permeability or vascular leakage, e.g., edema associated withbrain tumors, ascites associated with malignancies, Meigs' syndrome,lung inflammation, nephrotic syndrome, pericardial effusion, pleuraleffusion, permeability associated with cardiovascular diseases such asthe condition following myocardial infarctions and strokes and the like.

Other angiogenesis-dependent diseases according to this inventioninclude angiofibroma (abnormal blood of vessels which are prone tobleeding), neovascular glaucoma (growth of blood vessels in the eye),arteriovenous malformations (abnormal communication between arteries andveins), nonunion fractures (fractures that will not heal),atherosclerotic plaques (hardening of the arteries), pyogenic granuloma(common skin lesion composed of blood vessels), scleroderma (a form ofconnective tissue disease), hemangioma (tumor composed of bloodvessels), trachoma (leading cause of blindness in the third world),hemophilic joints, vascular adhesions and hypertrophic scars (abnormalscar formation).

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures. Those in need of treatment include those alreadywith the disorder as well as those in which the disorder is to beprevented.

The terms “recurrence,” “relapse” or “relapsed” refers to the return ofa cancer or disease after clinical assessment of the disappearance ofdisease. A diagnosis of distant metastasis or local recurrence can beconsidered a relapse.

The term “refractory” or “resistant” refers to a cancer or disease thathas not responded to treatment.

The term “adjuvant therapy” refers to treatment given after the primarytherapy, usually surgery. Adjuvant therapy for cancer or disease mayinclude immune therapy, chemotherapy, radiation therapy or hormonetherapy.

The term “maintenance therapy” refers to scheduled retreatment that isgiven to help maintain a previous treatment's effects. Maintenancetherapy is often given to help keep cancer in remission or prolong aresponse to a specific therapy regardless of disease progression.

The term “invasive cancer” refers to cancer that has spread beyond thelayer of tissue in which it started into the normal surrounding tissues.Invasive cancers may or may not be metastatic.

The term “non-invasive cancer” refers to a very early cancer or a cancerthat has not spread beyond the tissue of origin.

The term “progression-free survival” in oncology refers to the length oftime during and after treatment that a cancer does not grow.Progression-free survival includes the amount of time patients haveexperienced a complete response or a partial response, as well as theamount of time patients have experienced stable disease.

The term “progressive disease” in oncology can refer to a tumor growthof more than 20 percent since treatment began—either due to an increasein mass or a spread in the tumor.

A “disorder” is any condition that would benefit from treatment with theantibody. For example, mammals who suffer from or need prophylaxisagainst abnormal angiogenesis (excessive, inappropriate or uncontrolledangiogenesis) or abnormal vascular permeability or leakage. Thisincludes chronic and acute disorders or diseases including thosepathological conditions which predispose the mammal to the disorder inquestion. Non-limiting examples of disorders to be treated hereininclude malignant and benign tumors; non-leukemias and lymphoidmalignancies; neuronal, glial, astrocytal, hypothalamic and otherglandular, macrophagal, epithelial, stromal and blastocoelic disorders;and inflammatory, angiogenic and immunologic disorders.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include but are not limitedto, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include squamous cell cancer,glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladdercancer, hepatoma, breast cancer, colon cancer, colorectal cancer,endometrial carcinoma, salivary gland carcinoma, kidney cancer, renalcancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma, head and neck cancer, rectal cancer, colorectal cancer, lungcancer including small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung and squamous carcinoma of the lung, squamouscell cancer (e.g. epithelial squamous cell cancer), prostate cancer,cancer of the peritoneum, hepatocellular cancer, gastric or stomachcancer including gastrointestinal cancer, pancreatic cancer,glioblastoma, retinoblastoma, astrocytoma, thecomas, arrhenoblastomas,hepatoma, hematologic malignancies including non-Hodgkins lymphoma(NHL), multiple myeloma and acute hematologic malignancies, endometrialor uterine carcinoma, endometriosis, fibrosarcomas, choriocarcinoma,salivary gland carcinoma, vulval cancer, thyroid cancer, esophagealcarcinomas, hepatic carcinoma, anal carcinoma, penile carcinoma,nasopharyngeal carcinoma, laryngeal carcinomas, Kaposi's sarcoma,melanoma, skin carcinomas, Schwannoma, oligodendroglioma,neuroblastomas, rhabdomyosarcoma, osteogenic sarcoma, leiomyosarcomas,urinary tract carcinomas, thyroid carcinomas, Wilm's tumor, as well asB-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma(NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL;intermediate grade diffuse NHL; high grade immunoblastic NHL; high gradelymphoblastic NHL; high grade small non-cleaved cell NHL; bulky diseaseNHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom'sMacroglobulinemia); chronic lymphocytic leukemia (CLL); acutelymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblasticleukemia; and post-transplant lymphoproliferative disorder (PTLD), aswell as abnormal vascular proliferation associated with phakomatoses,and Meigs' syndrome.

“Tumor”, as used herein, refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues.

The term “anti-neoplastic composition” or “anti-neoplastic agent” refersto a composition useful in treating cancer comprising at least oneactive therapeutic agent, e.g., “anti-cancer agent.” Examples oftherapeutic agents (anti-cancer agents) include, but are limited to,e.g., chemotherapeutic agents, growth inhibitory agents, cytotoxicagents, agents used in radiation therapy, anti-angiogenesis agents,apoptotic agents, anti-tubulin agents, and other-agents to treat cancer,such as anti-HER-2 antibodies, anti-CD20 antibodies, an epidermal growthfactor receptor (EGFR) antagonist (e.g., a tyrosine kinase inhibitor),HER1/EGFR inhibitor (e.g., erlotinib (Tarceva™), platelet derived growthfactor inhibitors (e.g., Gleevec™ (Imatinib Mesylate)), a COX-2inhibitor (e.g., celecoxib), interferons, cytokines, antagonists (e.g.,neutralizing antibodies) that bind to one or more of the followingtargets ErbB2, ErbB3, ErbB4, PDGFR-beta, BAFF, BR3, APRIL, BCMA or VEGFreceptor(s), TRAIL/Apo2, and other bioactive and organic chemicalagents, etc. Combinations thereof are also contemplated in thisinvention.

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth or proliferation of a cell in vitroand/or in vivo. Thus, the growth inhibitory agent may be one whichsignificantly reduces the percentage of cells in S phase. Examples ofgrowth inhibitory agents include agents that block cell cycleprogression (at a place other than S phase), such as agents that induceG1 arrest and M-phase arrest. Classical M-phase blockers include thevincas (vincristine and vinblastine), TAXOL®, and topo II inhibitorssuch as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin.Those agents that arrest G1 also spill over into S-phase arrest, forexample, DNA alkylating agents such as tamoxifen, prednisone,dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil,and ara-C. Further information can be found in The Molecular Basis ofCancer, Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycleregulation, oncogenes, and antineoplastic drugs” by Murakami et al. (WBSaunders: Philadelphia, 1995), especially p. 13.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes e.g., I¹³¹,I¹²⁵, Y⁹⁰ and Re¹⁸⁶), chemotherapeutic agents, and toxins such asenzymatically active toxins of bacterial, fungal, plant or animalorigin, or fragments thereof.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents include is achemical compound useful in the treatment of cancer. Examples ofchemotherapeutic agents include alkylating agents such as thiotepa andCYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan,improsulfan and piposulfan; aziridines such as benzodopa, carboquone,meturedopa, and uredopa; ethylenimines and methylamelamines includingaltretamine, triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gamma11 and calicheamicinomega11 (see, e.g., Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994));dynemicin, including dynemicin A; bisphosphonates, such as clodronate;an esperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antiobiotic chromophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN®doxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL®paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine;NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate; daunomycin;aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11)(including the treatment regimen of irinotecan with 5-FU andleucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine(DMFO); retinoids such as retinoic acid; capecitabine; combretastatin;leucovorin (LV); oxaliplatin, including the oxaliplatin treatmentregimen (FOLFOX); inhibitors of PKC-alpha, Raf, H-Ras and EGFR (e.g.,erlotinib (Tarceva™)) that reduce cell proliferation andpharmaceutically acceptable salts, acids or derivatives of any of theabove.

Chemotherapeutic agents also include anti-hormonal agents that act toregulate or inhibit hormone action on tumors such as anti-estrogens andselective estrogen receptor modulators (SERMs), including, for example,tamoxifen (including NOLVADEX® tamoxifen), raloxifene, droloxifene,4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, andFARESTON• toremifene; aromatase inhibitors that inhibit the enzymearomatase, which regulates estrogen production in the adrenal glands,such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE®megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole,RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole; andanti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide,and goserelin; as well as troxacitabine (a 1,3-dioxolane nucleosidecytosine analog); antisense oligonucleotides, particularly those whichinhibit expression of genes in signaling pathways implicated in abherantcell proliferation, such as, for example, PKC-alpha, Raf and H-Ras;ribozymes such as a VEGF expression inhibitor (e.g., ANGIOZYME®ribozyme) and a HER2 expression inhibitor; vaccines such as gene therapyvaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, andVAXID® vaccine; PROLEUKIN® rIL-2; LURTOTECAN® topoisomerase 1 inhibitor;ABARELIX® rmRH; Vinorelbine and Esperamicins (see U.S. Pat. No.4,675,187), and pharmaceutically acceptable salts, acids or derivativesof any of the above.

The term “prodrug” as used in this application refers to a precursor orderivative form of a pharmaceutically active substance (e.g., smallmolecule) that is less cytotoxic to diseased cells compared to theparent drug and is capable of being enzymatically activated or convertedinto the more active parent form. See, e.g., Wilman, “Prodrugs in CancerChemotherapy” Biochemical Society Transactions, 14, pp. 375-382, 615thMeeting Belfast (1986) and Stella et al., “Prodrugs: A Chemical Approachto Targeted Drug Delivery,” Directed Drug Delivery, Borchardt et al.,(ed.), pp. 247-267, Humana Press (1985). The prodrugs of this inventioninclude, but are not limited to, phosphate-containing prodrugs,thiophosphate-containing prodrugs, sulfate-containing prodrugs,peptide-containing prodrugs, D-amino acid-modified prodrugs,glycosylated prodrugs, β-lactam-containing prodrugs, optionallysubstituted phenoxyacetamide-containing prodrugs or optionallysubstituted phenylacetamide-containing prodrugs, 5-fluorocytosine andother 5-fluorouridine prodrugs which can be converted into the moreactive cytotoxic free drug. Examples of cytotoxic drugs that can bederivatized into a prodrug form for use in this invention include, butare not limited to, those chemotherapeutic agents described above.

“Isolated,” when used to describe the various polypeptides disclosedherein, means polypeptide that has been identified and separated and/orrecovered from a cell or cell culture from which it was expressed.Contaminant components of its natural environment are materials thatwould typically interfere with diagnostic or therapeutic uses for thepolypeptide, and can include enzymes, hormones, and other proteinaceousor non-proteinaceous solutes. In preferred embodiments, the polypeptidewill be purified (1) to a degree sufficient to obtain at least 15residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (2) to homogeneity by SDS-PAGE undernon-reducing or reducing conditions using Coomassic blue or, preferably,silver stain. Isolated polypeptide includes polypeptide in situ withinrecombinant cells, since at least one component of the polypeptidenatural environment will not be present. Ordinarily, however, isolatedpolypeptide will be prepared by at least one purification step.

An “isolated” polypeptide-encoding nucleic acid or otherpolypeptide-encoding nucleic acid is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe polypeptide-encoding nucleic acid. An isolated polypeptide-encodingnucleic acid molecule is other than in the form or setting in which itis found in nature. Isolated polypeptide-encoding nucleic acid moleculestherefore are distinguished from the specific polypeptide-encodingnucleic acid molecule as it exists in natural cells. However, anisolated polypeptide-encoding nucleic acid molecule includespolypeptide-encoding nucleic acid molecules contained in cells thatordinarily express the polypeptide where, for example, the nucleic acidmolecule is in a chromosomal location different from that of naturalcells.

The term “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

“Stringent conditions” or “high stringency conditions”, as definedherein, can be identified by those that: (1) employ low ionic strengthand high temperature for washing, for example 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.;(2) employ during hybridization a denaturing agent, such as formamide,for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM sodium chloride, 75 mM sodium citrate at 42 C; or (3)overnight hybridization in a solution that employs 50% formamide, 5×SSC(0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8),0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon spermDNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with a 10minute wash at 42° C. in 0.2×SSC (sodium chloride/sodium citrate)followed by a 10 minute high-stringency wash consisting of 0.1×SSCcontaining EDTA at 55° C.

The amino acid sequences described herein are contiguous amino acidsequences unless otherwise specified.

As used herein, the term “immunoadhesin” designates antibody-likemolecules that combine the binding specificity of a heterologous protein(an “adhesin”) with the effector functions of immunoglobulin constantdomains. Structurally, the immunoadhesins comprise a fusion of an aminoacid sequence with the desired binding specificity that is other thanthe antigen recognition and binding site of an antibody (i.e., is“heterologous”), and an immunoglobulin constant domain sequence. Theadhesin part of an immunoadhesin molecule typically is a contiguousamino acid sequence comprising at least the binding site of a receptoror a ligand—such as a VEGFR or a fibronectin ligand. The immunoglobulinconstant domain sequence in the immunoadhesin can be obtained from anyimmunoglobulin, such as IgG-1, IgG-2, IgG-3, or IgG-4 subtypes, IgA(including IgA-1 and IgA-2), IgE, IgD, or IgM. Peptibodies, which oftencomprise a sequence derived from phage display selection of sequencesthat specifically bind a target fused to an Fc portion of animmunoglobulin, can be considered immunadhesins herein.

The term “antibody” is used in the broadest sense and specificallycovers, for example, single monoclonal antibodies (including agonist,antagonist, and neutralizing antibodies), antibody compositions withpolyepitopic specificity, polyclonal antibodies, single chainanti-antibodies, and fragments of antibodies (see below) as long as theyspecifically bind a native polypeptide and/or exhibit a biologicalactivity or immunological activity of this invention. According to oneembodiment, the antibody binds to an oligomeric form of a targetprotein, e.g., a trimeric form. According to another embodiment, theantibody specifically binds to a protein, which binding can be inhibitedby a monoclonal antibody of this invention (e.g., a deposited antibodyof this invention, etc.). The phrase “functional fragment or analog” ofan antibody is a compound having a qualitative biological activity incommon with an antibody to which it is being referred. For example, afunctional fragment or analog of an antibody of this invention can beone which can specifically bind to VEGF or α5β1. In one embodiment, theantibody can prevent or substantially reduce the ability of a VEGF toinduce cell proliferation.

An “isolated antibody” is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and can include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

The basic 4-chain antibody unit is a heterotetrameric glycoproteincomposed of two identical light (L) chains and two identical heavy (H)chains (an IgM antibody consists of 5 of the basic heterotetramer unitalong with an additional polypeptide called J chain, and thereforecontain 10 antigen binding sites, while secreted IgA antibodies canpolymerize to form polyvalent assemblages comprising 2-5 of the basic4-chain units along with J chain). In the case of IgGs, the 4-chain unitis generally about 150,000 daltons. Each L chain is linked to a H chainby one covalent disulfide bond, while the two H chains are linked toeach other by one or more disulfide bonds depending on the H chainisotype. Each H and L chain also has regularly spaced intrachaindisulfide bridges. Each H chain has at the N-terminus, a variable domain(V_(H)) followed by three constant domains (C_(H)) for each of the α andγ chains and four C_(H) domains for μ and ε isotypes. Each L chain hasat the N-terminus, a variable domain (V_(L)) followed by a constantdomain (C_(L)) at its other end. The V_(L) is aligned with the V_(H) andthe C_(L) is aligned with the first constant domain of the heavy chain(C_(H)1). Particular amino acid residues are believed to form aninterface between the light chain and heavy chain variable domains. Thepairing of a V_(H) and V_(L) together forms a single antigen-bindingsite. For the structure and properties of the different classes ofantibodies, see, e.g., BASIC AND CLINICAL IMMUNOLOGY, 8th edition,Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton& Lange, Norwalk, Conn., 1994, page 71 and Chapter 6.

The L chain from any vertebrate species can be assigned to one of twoclearly distinct types, called kappa and lambda, based on the amino acidsequences of their constant domains. Depending on the amino acidsequence of the constant domain of their heavy chains (C_(H)),immunoglobulins can be assigned to different classes or isotypes. Thereare five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, havingheavy chains designated α, δ, γ, ε, and μ, respectively. The γ and αclasses are further divided into subclasses on the basis of relativelyminor differences in C_(H) sequence and function, e.g., humans expressthe following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

The term “variable” refers to the fact that certain segments of thevariable domains differ extensively in sequence among antibodies. The Vdomain mediates antigen binding and define specificity of a particularantibody for its particular antigen. However, the variability is notevenly distributed across the 110-amino acid span of the variabledomains. Instead, the V regions consist of relatively invariantstretches called framework regions (FRs) of 15-30 amino acids separatedby shorter regions of extreme variability called “hypervariable regions”that are each 9-12 amino acids long. The variable domains of nativeheavy and light chains each comprise four FRs, largely adopting abeta-sheet configuration, connected by three hypervariable regions,which form loops connecting, and in some cases forming part of, thebeta-sheet structure. The hypervariable regions in each chain are heldtogether in close proximity by the FRs and, with the hypervariableregions from the other chain, contribute to the formation of theantigen-binding site of antibodies (see Kabat et al., SEQUENCES OFPROTEINS OF IMMUNOLOGICAL INTEREST, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). The constantdomains are not involved directly in binding an antibody to an antigen,but exhibit various effector functions, such as participation of theantibody in antibody dependent cellular cytotoxicity (ADCC).

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen-binding.The hypervariable region generally comprises amino acid residues from a“complementarity determining region” or “CDR” (e.g. around aboutresidues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the V_(L), and aroundabout 31-35B (H1), 50-65 (H2) and 95-102 (H3) in the V_(H) (in oneembodiment, H1 is around about 31-35); Kabat et al., supra and/or thoseresidues from a “hypervariable loop” (e.g. residues 26-32 (L1), 50-52(L2) and 91-96 (L3) in the V_(L), and 26-32 (H1), 53-55 (H2) and 96-101(H3) in the V_(H); Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).HVR regions in this invention include positions: 24-36 (LHVR1), 46-56(LHVR2), and 89-97 (LHVR3) of the light chain variable domain and 26-35(HHVR1), 47-65 (HHVR2) and 93-102 (HHVR3) of the heavy chain variabledomain

(Kabat numbering system, Kabat et al., supra 1991)

Throughout the present specification and claims, the Kabat numberingsystem is generally used when referring to a residue in the variabledomain (approximately, residues 1-107 of the light chain and residues1-113 of the heavy chain) (e.g, Kabat et al., supra (1991)). The “EUnumbering system” or “EU index” is generally used when referring to aresidue in an immunoglobulin heavy chain constant region (e.g., the EUindex reported in Kabat et al., supra (1991) expressly incorporatedherein by reference). Unless stated otherwise herein, references toresidues numbers in the variable domain of antibodies means residuenumbering by the Kabat numbering system. Unless stated otherwise herein,references to residue numbers in the constant domain of antibodies meansresidue numbering by the EU numbering system.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that can be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations which include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the antigen. In addition totheir specificity, the monoclonal antibodies are advantageous in thatthey can be synthesized uncontaminated by other antibodies. The modifier“monoclonal” is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies useful in the present invention can be prepared by thehybridoma methodology first described by Kohler et al., Nature, 256:495(1975), or can be made using recombinant DNA methods in bacterial,eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567).The “monoclonal antibodies” can also be isolated from phage antibodylibraries using the techniques described in Clackson et al., Nature,352:624-628 (1991), Marks et al., J. Mol. Biol., 222:581-597 (1991) orusing the methods set forth in the Examples below.

The monoclonal antibodies herein include “chimeric” antibodies in whicha portion of the heavy and/or light chain is identical with orhomologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular antibody class orsubclass, while the remainder of the chain(s) is identical with orhomologous to corresponding sequences in antibodies derived from anotherspecies or belonging to another antibody class or subclass, as well asfragments of such antibodies, so long as they exhibit a biologicalactivity of this invention (see U.S. Pat. No. 4,816,567; and Morrison etal., PNAS USA, 81:6851-6855 (1984)). Chimeric antibodies of interestherein include “primatized” antibodies comprising variable domainantigen-binding sequences derived from a non-human primate (e.g. OldWorld Monkey, Ape etc), and human constant region sequences.

An “intact” antibody is one which comprises an antigen-binding site aswell as a C_(L) and at least heavy chain constant domains, C_(H)1,C_(H)2 and C_(H)3. The constant domains can be native sequence constantdomains (e.g. human native sequence constant domains) or amino acidsequence variant thereof. Preferably, the intact antibody has one ormore effector functions.

“Antibody fragments” comprise a portion of an intact antibody,preferably the antigen binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, andFv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870,Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062 [1995]);single-chain antibody molecules; and multispecific antibodies formedfrom antibody fragments.

The expression “linear antibodies” generally refers to the antibodiesdescribed in Zapata et al., Protein Eng., 8(10):1057-1062 (1995).Briefly, these antibodies comprise a pair of tandem Fd segments(VH-CH1-VH-CH1) which, together with complementary light chainpolypeptides, form a pair of antigen binding regions. Linear antibodiescan be bispecific or monospecific.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, and a residual “Fc” fragment, adesignation reflecting the ability to crystallize readily. The Fabfragment consists of an entire L chain along with the variable regiondomain of the H chain (V_(H)), and the first constant domain of oneheavy chain (C_(H)1). Each Fab fragment is monovalent with respect toantigen binding, i.e., it has a single antigen-binding site. Pepsintreatment of an antibody yields a single large F(ab′)₂ fragment whichroughly corresponds to two disulfide linked Fab fragments havingdivalent antigen-binding activity and is still capable of cross-linkingantigen. Fab′ fragments differ from Fab fragments by having additionalfew residues at the carboxy terminus of the C_(H)1 domain including oneor more cysteines from the antibody hinge region. Fab′-SH is thedesignation herein for Fab′ in which the cysteine residue(s) of theconstant domains bear a free thiol group. F(ab′)₂ antibody fragmentsoriginally were produced as pairs of Fab′ fragments which have hingecysteines between them. Other chemical couplings of antibody fragmentsare also known.

The Fc fragment comprises the carboxy-terminal portions of both H chainsheld together by disulfides. The effector functions of antibodies aredetermined by sequences in the Fc region, which region is also the partrecognized by Fc receptors (FcR) found on certain types of cells.

A “variant Fc region” comprises an amino acid sequence which differsfrom that of a native sequence Fc region by virtue of at least one“amino acid modification” as herein defined. Preferably, the variant Fcregion has at least one amino acid substitution compared to a nativesequence Fc region or to the Fc region of a parent polypeptide, e.g.from about one to about ten amino acid substitutions, and preferablyfrom about one to about five amino acid substitutions in a nativesequence Fc region or in the Fc region of the parent polypeptide. In oneembodiment, the variant Fc region herein will possess at least about 80%homology, at least about 85% homology, at least about 90% homology, atleast about 95% homology or at least about 99% homology with a nativesequence Fc region. According to another embodiment, the variant Fcregion herein will possess at least about 80% homology, at least about85% homology, at least about 90% homology, at least about 95% homologyor at least about 99% homology with an Fc region of a parentpolypeptide.

The term “Fc region-comprising polypeptide” refers to a polypeptide,such as an antibody or immunoadhesin (see definitions below), whichcomprises an Fc region. The C-terminal lysine (residue 447 according tothe EU numbering system) of the Fc region may be removed, for example,during purification of the polypeptide or by recombinantly engineeringthe nucleic acid encoding the polypeptide. Accordingly, a compositioncomprising polypeptides, including antibodies, having an Fc regionaccording to this invention can comprise polypeptides populations withall K447 residues removed, polypeptide populations with no K447 residuesremoved or polypeptide populations having a mixture of polypeptides withand without the K447 residue.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This fragment consists of a dimerof one heavy- and one light-chain variable region domain in tight,non-covalent association. From the folding of these two domains emanatesix hypervariable loops (3 loops each from the H and L chain) thatcontribute the amino acid residues for antigen binding and conferantigen binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

“Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibodyfragments that comprise the V_(H) and V_(L) antibody domains connectedinto a single polypeptide chain. Preferably, the sFv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains whichenables the sFv to form the desired structure for antigen binding. For areview of sFv, see Pluckthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994); Borrebaeck 1995, infra.

The term “diabodies” refers to small antibody fragments prepared byconstructing sFv fragments (see preceding paragraph) with short linkers(about 5-10 residues) between the V_(H) and V_(L) domains such thatinter-chain but not intra-chain pairing of the V domains is achieved,resulting in a bivalent fragment, i.e., fragment having twoantigen-binding sites. Bispecific diabodies are heterodimers of two“crossover” sFv fragments in which the V_(H) and V_(L) domains of thetwo antibodies are present on different polypeptide chains. Diabodiesare described more fully in, for example, EP 404,097; WO 93/11161; andHollinger et al., PNAS USA, 90:6444-6448 (1993).

“Humanized” forms of non-human (e.g., rodent) antibodies are chimericantibodies that contain minimal sequence derived from the non-humanantibody. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or non-human primate having the desired antibodyspecificity, affinity, and capability. In some instances, frameworkregion (FR) residues of the human immunoglobulin are replaced bycorresponding non-human residues. Furthermore, humanized antibodies cancomprise residues that are not found in the recipient antibody or in thedonor antibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, sec Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992).

A “species-dependent antibody” is an antibody which has a strongerbinding affinity for an antigen from a first mammalian species than ithas for a homologue of that antigen from a second mammalian species.Normally, the species-dependent antibody “bind specifically” to a humanantigen (i.e., has a binding affinity (Kd) value of no more than about1×10⁻⁷ M, no more than about 1×10⁻⁸ or no more than about 1×10⁻⁹ M) buthas a binding affinity for a homologue of the antigen from a secondnon-human mammalian species which is at least about 50 fold, or at leastabout 500 fold, or at least about 1000 fold, weaker than its bindingaffinity for the human antigen. The species-dependent antibody can be ofany of the various types of antibodies as defined above, but preferablyis a humanized or human antibody.

In such embodiments, the extent of binding of the polypeptide, antibody,antagonist or composition to a “non-target” protein will be less thanabout 10% of the binding of the polypeptide, antibody, antagonist orcomposition to its particular target protein as determined byfluorescence activated cell sorting (FACS) analysis orradioimmunoprecipitation (RIA). With regard to the binding of apolypeptide, antibody, antagonist or composition to a target molecule,the term “specific binding” or “specifically binds to” or is “specificfor” a particular polypeptide or an epitope on a particular polypeptidetarget means binding that is measurably different from a non-specificinteraction. Specific binding can be measured, for example, bydetermining binding of a molecule compared to binding of a controlmolecule, which generally is a molecule of similar structure that doesnot have binding activity. For example, specific binding can bedetermined by competition with a control molecule that is similar to thetarget, for example, an excess of non-labeled target. In this case,specific binding is indicated if the binding of the labeled target to aprobe is competitively inhibited by excess unlabeled target. The term“specific binding” or “specifically binds to” or is “specific for” aparticular polypeptide or an epitope on a particular polypeptide targetas used herein can be exhibited, for example, by a molecule having a Kdfor the target of at least about 10⁻⁴ M, at least about 10⁻⁵ M, at leastabout 10⁻⁶ M, at least about 10⁻⁷ M, at least about 10⁻⁸ M, at leastabout 10⁻⁹ M, alternatively at least about 10⁻¹⁰ M, at least about 10⁻¹¹M, at least about 10⁻¹² M, or greater. In one embodiment, the term“specific binding” refers to binding where a molecule binds to aparticular polypeptide or epitope on a particular polypeptide withoutsubstantially binding to any other polypeptide or polypeptide epitope.

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody, and vary with the antibodyisotype. Examples of antibody effector functions include: C1q bindingand complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors; and B cell activation. A “nativesequence Fc region” comprises an amino acid sequence identical to theamino acid sequence of an Fc region found in nature. Examples of Fcsequences are described in, for example, but not limited to, Kabat etal., supra (1991)).

“Percent (%) amino acid sequence identity” or “homology” with respect tothe polypeptide and antibody sequences identified herein is defined asthe percentage of amino acid residues in a candidate sequence that areidentical with the amino acid residues in the polypeptide beingcompared, after aligning the sequences considering any conservativesubstitutions as part of the sequence identity. Alignment for purposesof determining percent amino acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN orMegalign (DNASTAR) software. Those skilled in the art can determineappropriate parameters for measuring alignment, including any algorithmsneeded to achieve maximal alignment over the full length of thesequences being compared. For purposes herein, however, % amino acidsequence identity values are generated using the sequence comparisoncomputer program ALIGN-2. The ALIGN-2 sequence comparison computerprogram was authored by Genentech, Inc. and the source code has beenfiled with user documentation in the U.S. Copyright Office, WashingtonD.C., 20559, where it is registered under U.S. Copyright RegistrationNo. TXU510087. The ALIGN-2 program is publicly available throughGenentech, Inc., South San Francisco, Calif. The ALIGN-2 program shouldbe compiled for use on a UNIX operating system, preferably digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

The terms “Fc receptor” or “FcR” are used to describe a receptor thatbinds to the Fc region of an antibody. In one embodiment, an FcR of thisinvention is one that binds an IgG antibody (a gamma receptor) andincludes receptors of the FcγRI, FcγRII, and FcγRIII subclasses,including allelic variants and alternatively spliced forms of thesereceptors. FcγRII receptors include FcγRIIA (an “activating receptor”)and FcγRIIB (an “inhibiting receptor”), which have similar amino acidsequences that differ primarily in the cytoplasmic domains thereof.Activating receptor FcγRIIA contains an immunoreceptor tyrosine-basedactivation motif (ITAM) in its cytoplasmic domain Inhibiting receptorFcγRIIB contains an immunoreceptor tyrosine-based inhibition motif(ITIM) in its cytoplasmic domain. (see review M. in Daëron, Annu. Rev.Immunol. 15:203-234 (1997)). The term includes allotypes, such asFcγRIIIA allotypes: FcγRIIIA-Phe158, FcγRIIIA-Val158, FcγRIIA-R131and/or FcγRIIA-H131. FcRs are reviewed in Ravetch and Kinet, Annu. Rev.Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); andde Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs,including those to be identified in the future, are encompassed by theterm “FcR” herein. The term also includes the neonatal receptor, FcRn,which is responsible for the transfer of maternal IgGs to the fetus(Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol.24:249 (1994)).

The term “FcRn” refers to the neonatal Fc receptor (FcRn). FcRn isstructurally similar to major histocompatibility complex (MHC) andconsists of an α-chain noncovalently bound to β2-microglobulin. Themultiple functions of the neonatal Fc receptor FcRn are reviewed inGhetie and Ward (2000) Annu. Rev. Immunol. 18, 739-766. FcRn plays arole in the passive delivery of immunoglobulin IgGs from mother to youngand the regulation of serum IgG levels. FcRn can act as a salvagereceptor, binding and transporting pinocytosed IgGs in intact form bothwithin and across cells, and rescuing them from a default degradativepathway.

WO 00/42072 (Presta) and Shields et al. J. Biol. Chem. 9(2): 6591-6604(2001) describe antibody variants with improved or diminished binding toFcRs. The contents of those publications are specifically incorporatedherein by reference.

The “CH1 domain” of a human IgG Fc region (also referred to as “C1” of“H1” domain) usually extends from about amino acid 118 to about aminoacid 215 (EU numbering system).

“Hinge region” is generally defined as stretching from Glu216 to Pro230of human IgG1 (Burton, Molec. Immunol. 22:161-206 (1985)). Hinge regionsof other IgG isotypes may be aligned with the IgG1 sequence by placingthe first and last cysteine residues forming inter-heavy chain S—S bondsin the same positions.

The “lower hinge region” of an Fc region is normally defined as thestretch of residues immediately C-terminal to the hinge region, i.e.residues 233 to 239 of the Fc region. In previous reports, FcR bindingwas generally attributed to amino acid residues in the lower hingeregion of an IgG Fc region.

The “CH2 domain” of a human IgG Fc region (also referred to as “C2” of“H2” domain) usually extends from about amino acid 231 to about aminoacid 340. The CH2 domain is unique in that it is not closely paired withanother domain. Rather, two N-linked branched carbohydrate chains areinterposed between the two CH2 domains of an intact native IgG molecule.It has been speculated that the carbohydrate may provide a substitutefor the domain-domain pairing and help stabilize the CH2 domain. Burton,Molec. Immunol. 22:161-206 (1985).

The “CH3 domain” (also referred to as “C2” or “H3” domain) comprises thestretch of residues C-terminal to a CH2 domain in an Fc region (i.e.from about amino acid residue 341 to the C-terminal end of an antibodysequence, typically at amino acid residue 446 or 447 of an IgG).

A “functional Fc region” possesses an “effector function” of a nativesequence Fc region. Exemplary “effector functions” include C1q binding;complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor; BCR), etc.Such effector functions generally require the Fc region to be combinedwith a binding domain (e.g. an antibody variable domain) and can beassessed using various assays as herein disclosed, for example.

“C1q” is a polypeptide that includes a binding site for the Fc region ofan immunoglobulin. C1q together with two serine proteases, C1r and C1s,forms the complex C1, the first component of the complement dependentcytotoxicity (CDC) pathway. Human C1q can be purchased commerciallyfrom, e.g. Quidel, San Diego, Calif.

The term “binding domain” refers to the region of a polypeptide thatbinds to another molecule. In the case of an FcR, the binding domain cancomprise a portion of a polypeptide chain thereof (e.g. the alpha chainthereof) which is responsible for binding an Fc region. One usefulbinding domain is the extracellular domain of an FcR alpha chain.

An antibody or peptibody with a variant IgG Fc with “altered” FcRbinding affinity or ADCC activity is one which has either enhanced ordiminished FcR binding activity (e.g, FcγR or FcRn) and/or ADCC activitycompared to a parent polypeptide or to a polypeptide comprising a nativesequence Fc region. The variant Fc which “exhibits increased binding” toan FcR binds at least one FcR with higher affinity (e.g., lower apparentKd or IC50 value) than the parent polypeptide or a native sequence IgGFc. According to some embodiments, the improvement in binding comparedto a parent polypeptide is about 3 fold, preferably about 5, 10, 25, 50,60, 100, 150, 200, 250, 300, 350, 400, 450, or 500 fold, or about 25% to1000% improvement in binding. The polypeptide variant which “exhibitsdecreased binding” to an FcR, binds at least one FcR with lower affinity(e.g, higher apparent Kd or higher IC50 value) than a parentpolypeptide. The decrease in binding compared to a parent polypeptidemay be about 5%, 10%, 20%, 30%, 40%, 50%, 60%, or more decrease inbinding.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to aform of cytotoxicity in which secreted Ig bound to Fc receptors (FcRs)present on certain cytotoxic cells (e.g. Natural Killer (NK) cells,neutrophils, and macrophages) enable these cytotoxic effector cells tobind specifically to an antigen-bearing target cell and subsequentlykill the target cell with cytotoxins. The antibodies “arm” the cytotoxiccells and are absolutely required for such killing. The primary cellsfor mediating ADCC, NK cells, express FcγRIII only, whereas monocytesexpress FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cellsis summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev.Immunol 9:457-92 (1991). To assess ADCC activity of a molecule ofinterest, an in vitro ADCC assay, such as that described in U.S. Pat.No. 5,500,362 or 5,821,337 or in the Examples below may be performed.Useful effector cells for such assays include peripheral bloodmononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively,or additionally, ADCC activity of the molecule of interest may beassessed in vivo, e.g., in a animal model such as that disclosed inClynes et al. PNAS (USA) 95:652-656 (1998).

The polypeptide comprising a variant Fc region which “exhibits increasedADCC” or mediates antibody-dependent cell-mediated cytotoxicity (ADCC)in the presence of human effector cells more effectively than apolypeptide having wild type IgG Fc or a parent polypeptide is one whichin vitro or in vivo is substantially more effective at mediating ADCC,when the amounts of polypeptide with variant Fc region and thepolypeptide with wild type Fc region (or the parent polypeptide) in theassay are essentially the same. Generally, such variants will beidentified using the in vitro ADCC assay as herein disclosed, but otherassays or methods for determining ADCC activity, e.g. in an animal modeletc, are contemplated. In one embodiment, the preferred variant is fromabout 5 fold to about 100 fold, e.g. from about 25 to about 50 fold,more effective at mediating ADCC than the wild type Fc (or parentpolypeptide).

“Complement dependent cytotoxicity” or “CDC” refers to the lysis of atarget cell in the presence of complement. Activation of the classicalcomplement pathway is initiated by the binding of the first component ofthe complement system (C1q) to antibodies (of the appropriate subclass)which are bound to their cognate antigen. To assess complementactivation, a CDC assay, e.g. as described in Gazzano-Santoro et al., J.Immunol. Methods 202:163 (1996), may be performed.

Polypeptide variants with altered Fc region amino acid sequences andincreased or decreased C1q binding capability are described in U.S. Pat.No. 6,194,551 and WO 99/51642. The contents of those patent publicationsare specifically incorporated herein by reference. See, also, Idusogieet al. J. Immunol. 164: 4178-4184 (2000).

“Human effector cells” are leukocytes which express one or more FcRs andperform effector functions. According to one embodiment, the cellsexpress at least FcγRIII and perform ADCC effector function. Examples ofhuman leukocytes which mediate ADCC include peripheral blood mononuclearcells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cellsand neutrophils; with PBMCs and NK cells being preferred. The effectorcells may be isolated from a native source thereof, e.g. from blood orPBMCs as described herein.

Methods of measuring binding to FcRn are known (see, e.g., Ghetie 1997,Hinton 2004) as well as described in the Examples below. Binding tohuman FcRn in vivo and serum half life of human FcRn high affinitybinding polypeptides can be assayed, e.g, in transgenic mice ortransfected human cell lines expressing human FcRn, or in primatesadministered with the Fc variant polypeptides. In one embodiment,specifically the anti-α5β1 antibodies of the invention having a variantIgG Fc exhibits increased binding affinity for human FcRn over apolypeptide having wild-type IgG Fc, by at least 2 fold, at least 5fold, at least 10 fold, at least 50 fold, at least 60 fold, at least 70fold, at least 80 fold, at least 100 fold, at least 125 fold, at least150 fold. In a specific embodiment, the binding affinity for human FcRnis increased about 170 fold.

For binding affinity to FcRn, in one embodiment, the EC50 or apparent Kd(at pH 6.0) of the polypeptide is less than 1 uM, more preferably lessthan or equal to 100 nM, more preferably less than or equal to 10 nM. Inone embodiment, for increased binding affinity to FcγRIII (F158; i.e.low-affinity isotype) the EC50 or apparent Kd less is than or equal to10 nM, and for FcγRIII (V158; high-affinity isotype) the EC50 orapparent Kd is less than or equal to 3 nM. According to anotherembodiment, a reduction in binding of an antibody to a Fc receptorrelative to a control antibody (e.g., the Herceptin® antibody) may beconsidered significant relative to the control antibody if the ratio ofthe values of the absorbances at the midpoints of the test antibody andcontrol antibody binding curves (e.g,A_(450 nm(antibody))/A_(450 nm(control Ab))) is less than or equal to40%. According to another embodiment, an increase in binding of anantibody to a Fc receptor relative to a control antibody (e.g., theHerceptin® antibody) may be considered significant relative to thecontrol antibody if the ratio of the values of the absorbances at themidpoints of the test antibody and control antibody binding curves (e.g,A_(450 nm(antibody))/A_(450 nm(control Ab))) is greater than or equal to125%.

A “parent polypeptide” or “parent antibody” is a polypeptide or antibodycomprising an amino acid sequence from which the variant polypeptide orantibody arose and against which the variant polypeptide or antibody isbeing compared. Typically the parent polypeptide or parent antibodylacks one or more of the Fc region modifications disclosed herein anddiffers in effector function compared to a polypeptide variant as hereindisclosed. The parent polypeptide may comprise a native sequence Fcregion or an Fc region with pre-existing amino acid sequencemodifications (such as additions, deletions and/or substitutions).

Antibodies of this invention can be derived from phage display. As usedherein, “library” refers to a plurality of antibody or antibody fragmentsequences, or the nucleic acids that encode these sequences, thesequences being different in the combination of variant amino acids thatare introduced into these sequences according to the methods of theinvention.

“Phage display” is a technique by which variant polypeptides aredisplayed as fusion proteins to at least a portion of coat protein onthe surface of phage, e.g., filamentous phage, particles. A utility ofphage display lies in the fact that large libraries of randomizedprotein variants can be rapidly and efficiently sorted for thosesequences that bind to a target antigen with high affinity. Display ofpeptide and protein libraries on phage has been used for screeningmillions of polypeptides for ones with specific binding properties.Polyvalent phage display methods have been used for displaying smallrandom peptides and small proteins through fusions to either gene III orgene VIII of filamentous phage. Wells and Lowman, Curr. Opin. Struct.Biol., 3:355-362 (1992), and references cited therein. In a monovalentphage display, a protein or peptide library is fused to a gene III or aportion thereof, and expressed at low levels in the presence of wildtype gene III protein so that phage particles display one copy or noneof the fusion proteins. Avidity effects are reduced relative topolyvalent phage so that sorting is on the basis of intrinsic ligandaffinity, and phagemid vectors are used, which simplify DNAmanipulations. Lowman and Wells, Methods: A companion to Methods inEnzymology, 3:205-0216 (1991).

A “phagemid” is a plasmid vector having a bacterial origin ofreplication, e.g., Co1E1, and a copy of an intergenic region of abacteriophage. The phagemid may be used on any known bacteriophage,including filamentous bacteriophage and lambdoid bacteriophage. Theplasmid will also generally contain a selectable marker for antibioticresistance. Segments of DNA cloned into these vectors can be propagatedas plasmids. When cells harboring these vectors are provided with allgenes necessary for the production of phage particles, the mode ofreplication of the plasmid changes to rolling circle replication togenerate copies of one strand of the plasmid DNA and package phageparticles. The phagemid may form infectious or non-infectious phageparticles. This term includes phagemids which contain a phage coatprotein gene or fragment thereof linked to a heterologous polypeptidegene as a gene fusion such that the heterologous polypeptide isdisplayed on the surface of the phage particle.

The term “phage vector” means a double stranded replicative form of abacteriophage containing a heterologous gene and capable of replication.The phage vector has a phage origin of replication allowing phagereplication and phage particle formation. The phage is preferably afilamentous bacteriophage, such as an M13, f1, fd, Pf3 phage or aderivative thereof, or a lambdoid phage, such as lambda, 21, phi80,phi81, 82, 424, 434, etc., or a derivative thereof.

Covalent modifications of polypeptides such as peptibodies,immunoadesins, antibodies and short peptides are included within thescope of this invention. One type of covalent modification includesreacting targeted amino acid residues of a polypeptide with an organicderivatizing agent that is capable of reacting with selected side chainsor the N- or C-terminal residues of the polypeptide. Derivatization withbifunctional agents is useful, for instance, for crosslinking thepolypeptide to a water-insoluble support matrix or surface for use inthe method for purifying antibodies, and vice-versa. Commonly usedcrosslinking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane,glutaraldehyde, N-hydroxy-succinimide esters, for example, esters with4-azidosalicylic acid, homobifunctional imidoesters, includingdisuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate),bifunctional maleimides such as bis-N-maleimido-1,8-octane and agentssuch as methyl-3-[(p-azidophenyl)dithio]propioimidate.

Other modifications include deamidation of glutaminyl and asparaginylresidues to the corresponding glutamyl and aspartyl residues,respectively, hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl or threonyl residues, methylation of theα-amino groups of lysine, arginine, and histidine side chains [T. E.Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman &Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminalamine, and amidation of any C-terminal carboxyl group.

Other modifications include the conjugation of toxins to the antagonistssuch as maytansine and maytansinoids, calicheamicin and other cytotoxicagents.

Another type of covalent modification of the polypeptide compriseslinking the polypeptide to one of a variety of nonproteinaceouspolymers, e.g., polyethylene glycol (PEG), polypropylene glycol, orpolyoxyalkylenes, in the manner set forth in U.S. Pat. No. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

The polypeptide of the present invention can also be modified ifadvantageous in a way to form a chimeric molecule comprising thepolypeptide fused to another, heterologous polypeptide or amino acidsequence (e.g., immunoadhesins or peptibodies).

In one embodiment, such a chimeric molecule comprises a fusion of thepolypeptide with a protein transduction domain which targets thepolypeptide for delivery to various tissues and more particularly acrossthe brain blood barrier, using, for example, the protein transductiondomain of human immunodeficiency virus TAT protein (Schwarze et al.,1999, Science 285: 1569-72).

In another embodiment, such a chimeric molecule comprises a fusion ofthe polypeptide with a tag polypeptide which provides an epitope towhich an anti-tag antibody can selectively bind. The epitope tag isgenerally placed at the amino- or carboxyl-terminus of the polypeptide.The presence of such epitope-tagged forms of the polypeptide can bedetected using an antibody against the tag polypeptide. Also, provisionof the epitope tag enables the polypeptide to be readily purified byaffinity purification using an anti-tag antibody or another type ofaffinity matrix that binds to the epitope tag. Various tag polypeptidesand their respective antibodies are known in the art. Examples includepoly-histidine (poly-His) or poly-histidine-glycine (poly-His-gly) tags;the flu HA tag polypeptide and its antibody 12CA5 [Field et al., Mol.Cell. Biol., 8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10,G4, B7 and 9E10 antibodies thereto [Evan et al., Molecular and CellularBiology, 5:3610-3616 (1985)]; and the Herpes Simplex virus glycoproteinD (gD) tag and its antibody [Paborsky et al., Protein Engineering,3(6):547-553 (1990)]. Other tag polypeptides include the Flag-peptide[Hopp et al., BioTechnology, 6:1204-1210 (1988)]; the KT3 epitopepeptide [Martin et al., Science, 255:192-194 (1992)]; an α-tubulinepitope peptide [Skinner et al., J. Biol. Chem., 266:15163-15166(1991)]; and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al.,PNAS USA, 87:6393-6397 (1990)].

In an alternative embodiment, the chimeric molecule can comprise afusion of the polypeptide with an immunoglobulin or a particular regionof an immunoglobulin. For a bivalent form of the chimeric molecule(e.g., an “immunoadhesin”), such a fusion could be to the Fc region ofan IgG molecule. Ig fusions of this invention include polypeptides thatcomprise approximately or only residues 94-243, residues 33-53 orresidues 33-52 of human in place of at least one variable region withinan Ig molecule. In a particularly preferred embodiment, theimmunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge,CH1, CH2 and CH3 regions of an IgG1 molecule. For the production ofimmunoglobulin fusions see also, U.S. Pat. No. 5,428,130.

The invention provides methods and compositions for inhibiting orpreventing relapse tumor growth or relapse cancer cell growth. Invarious embodiments, a cancer is relapse tumor growth or relapse cancercell growth where the number of cancer cells has not been significantlyreduced, or has increased, or tumor size has not been significantlyreduced, or has increased, or fails any further reduction in size or innumber of cancer cells. The determination of whether the cancer cellsare relapse tumor growth or relapse cancer cell growth can be madeeither in vivo or in vitro by any method known in the art for assayingthe effectiveness of treatment on cancer cells. A tumor resistant toanti-VEGF treatment is an example of a relapse tumor growth.

An “effective amount” of a polypeptide, antibody, antagonist orcomposition as disclosed herein is an amount sufficient to carry out aspecifically stated purpose. An “effective amount” can be determinedempirically and by known methods relating to the stated purpose.

The term “therapeutically effective amount” refers to an amount of anantibody, polypeptide or antagonist of this invention effective to“treat” a disease or disorder in a mammal (aka patient). In the case ofcancer, the therapeutically effective amount of the drug can reduce thenumber of cancer cells; reduce the tumor size or weight; inhibit (i.e.,slow to some extent and preferably stop) cancer cell infiltration intoperipheral organs; inhibit (i.e., slow to some extent and preferablystop) tumor metastasis; inhibit, to some extent, tumor growth; and/orrelieve to some extent one or more of the symptoms associated with thecancer. To the extent the drug can prevent growth and/or kill existingcancer cells, it can be cytostatic and/or cytotoxic. In one embodiment,the therapeutically effective amount is a growth inhibitory amount. Inanother embodiment, the therapeutically effective amount is an amountthat extends the survival of a patient. In another embodiment, thetherapeutically effective amount is an amount that improves progressionfree survival of a patient.

In the case of wound healing, the term “effective amount” or“therapeutically effective amount” refers to an amount of a drugeffective to accelerate or improve wound healing in a subject. Atherapeutic dose is a dose which exhibits a therapeutic effect on thepatient and a sub-therapeutic dose is a dose which does not exhibit atherapeutic effect on the patient treated.

A “chronic wound” refers a wound that does not heal. See, e.g., Lazaruset al., Definitions and guidelines for assessment of wounds andevaluation of healing, Arch. Dermatol. 130:489-93 (1994). Chronic woundsinclude, but are not limited to, e.g., arterial ulcers, diabetic ulcers,pressure ulcers, venous ulcers, etc. An acute wound can develop into achronic wound. Acute wounds include, but are not limited to, woundscaused by, e.g., thermal injury, trauma, surgery, excision of extensiveskin cancer, deep fungal and bacterial infections, vasculitis,scleroderma, pemphigus, toxic epidermal necrolysis, etc. See, e.g.,Buford, Wound Healing and Pressure Sores, HealingWell.com, published on:Oct. 24, 2001. A “normal wound” refers a wound that undergoes normalwound healing repair.

A “growth inhibitory amount” of a polypeptide, antibody, antagonist orcomposition of this invention is an amount capable of inhibiting thegrowth of a cell, especially tumor, e.g., cancer cell, either in vitroor in vivo. A “growth inhibitory amount” of a polypeptide, antibody,antagonist or composition of this invention for purposes of inhibitingneoplastic cell growth can be determined empirically and by knownmethods or by examples provided herein.

A “cytotoxic amount” of a polypeptide, antibody, antagonist orcomposition of this invention is an amount capable of causing thedestruction of a cell, especially tumor, e.g., cancer cell, either invitro or in vivo. A “cytotoxic amount” of a polypeptide, antibody,antagonist or composition of this invention for purposes of inhibitingneoplastic cell growth can be determined empirically and by methodsknown in the art.

An “autoimmune disease” herein is a disease or disorder arising from anddirected against an individual's own tissues or a co-segregate ormanifestation thereof or resulting condition therefrom. Examples ofautoimmune diseases or disorders include, but are not limited toarthritis (rheumatoid arthritis such as acute arthritis, chronicrheumatoid arthritis, gouty arthritis, acute gouty arthritis, chronicinflammatory arthritis, degenerative arthritis, infectious arthritis,Lyme arthritis, proliferative arthritis, psoriatic arthritis, vertebralarthritis, and juvenile-onset rheumatoid arthritis, osteoarthritis,arthritis chronica progrediente, arthritis deformans, polyarthritischronica primaria, reactive arthritis, and ankylosing spondylitis),inflammatory hyperproliferative skin diseases, psoriasis such as plaquepsoriasis, gutatte psoriasis, pustular psoriasis, and psoriasis of thenails, dermatitis including contact dermatitis, chronic contactdermatitis, allergic dermatitis, allergic contact dermatitis, dermatitisherpetiformis, and atopic dermatitis, x-linked hyper IgM syndrome,urticaria such as chronic idiopathic urticaria, including chronicautoimmune urticaria, polymyositis/dermatomyositis, juveniledermatomyositis, toxic epidermal necrolysis, scleroderma (includingsystemic scleroderma), sclerosis such as systemic sclerosis, multiplesclerosis (MS) such as spino-optical MS, primary progressive MS, andrelapsing remitting MS, progressive systemic sclerosis, atherosclerosis,arteriosclerosis, sclerosis disseminate, and ataxic sclerosis,inflammatory bowel disease (IBD) (for example, Crohn's disease, colitissuch as ulcerative colitis, colitis ulcerosa, microscopic colitis,collagenous colitis, colitis polyposa, necrotizing enterocolitis, andtransmural colitis, and autoimmune inflammatory bowel disease), pyodermagangrenosum, erythema nodosum, primary sclerosing cholangitis,episcleritis), respiratory distress syndrome, including adult or acuterespiratory distress syndrome (ARDS), meningitis, inflammation of all orpart of the uvea, iritis, choroiditis, an autoimmune hematologicaldisorder, rheumatoid spondylitis, sudden hearing loss, IgE-mediateddiseases such as anaphylaxis and allergic and atopic rhinitis,encephalitis such as Rasmussen's encephalitis and limbic and/orbrainstem encephalitis, uveitis, such as anterior uveitis, acuteanterior uveitis, granulomatous uveitis, nongranulomatous uveitis,phacoantigenic uveitis, posterior uveitis, or autoimmune uveitis,glomerulonephritis (GN) with and without nephrotic syndrome such aschronic or acute glomerulonephritis such as primary GN, immune-mediatedGN, membranous GN (membranous nephropathy), idiopathic membranous GN,membranous proliferative GN (MPGN), including Type I and Type II, andrapidly progressive GN, allergic conditions, allergic reaction, eczemaincluding allergic or atopic eczema, asthma such as asthma bronchiale,bronchial asthma, and auto-immune asthma, conditions involvinginfiltration of T cells and chronic inflammatory responses, chronicpulmonary inflammatory disease, autoimmune myocarditis, leukocyteadhesion deficiency, systemic lupus erythematosus (SLE) or systemiclupus erythematodes such as cutaneous SLE, subacute cutaneous lupuserythematosus, neonatal lupus syndrome (NLE), lupus erythematosusdisseminatus, lupus (including nephritis, cerebritis, pediatric,non-renal, discoid, alopecia), juvenile onset (Type I) diabetesmellitus, including pediatric insulin-dependent diabetes mellitus(IDDM), adult onset diabetes mellitus (Type II diabetes), autoimmunediabetes, idiopathic diabetes insipidus, immune responses associatedwith acute and delayed hypersensitivity mediated by cytokines andT-lymphocytes, tuberculosis, sarcoidosis, granulomatosis includinglymphomatoid granulomatosis, Wegener's granulomatosis, agranulocytosis,vasculitides, including vasculitis (including large vessel vasculitis(including polymyalgia rheumatica and giant cell (Takayasu's)arteritis), medium vessel vasculitis (including Kawasaki's disease andpolyarteritis nodosa), microscopic polyarteritis, CNS vasculitis,necrotizing, cutaneous, or hypersensitivity vasculitis, systemicnecrotizing vasculitis, and ANCA-associated vasculitis, such asChurg-Strauss vasculitis or syndrome (CSS)), temporal arteritis,aplastic anemia, autoimmune aplastic anemia, Coombs positive anemia,Diamond Blackfan anemia, hemolytic anemia or immune hemolytic anemiaincluding autoimmune hemolytic anemia (AIHA), pernicious anemia (anemiapemiciosa), Addison's disease, pure red cell anemia or aplasia (PRCA),Factor VIII deficiency, hemophilia A, autoimmune neutropenia,pancytopenia, leukopenia, diseases involving leukocyte diapedesis, CNSinflammatory disorders, multiple organ injury syndrome such as thosesecondary to septicemia, trauma or hemorrhage, antigen-antibodycomplex-mediated diseases, anti-glomerular basement membrane disease,anti-phospholipid antibody syndrome, allergic neuritis, Bechet's orBehcet's disease, Castleman's syndrome, Goodpasture's syndrome,Reynaud's syndrome, Sjogren's syndrome, Stevens-Johnson syndrome,pemphigoid such as pemphigoid bullous and skin pemphigoid, pemphigus(including pemphigus vulgaris, pemphigus foliaceus, pemphigusmucus-membrane pemphigoid, and pemphigus erythematosus), autoimmunepolyendocrinopathies, Reiter's disease or syndrome, immune complexnephritis, antibody-mediated nephritis, chronic neuropathy such as IgMpolyneuropathies or IgM-mediated neuropathy, thrombocytopenia (asdeveloped by myocardial infarction patients, for example), includingthrombotic thrombocytopenic purpura (TTP) and autoimmune orimmune-mediated thrombocytopenia such as idiopathic thrombocytopenicpurpura (ITP) including chronic or acute ITP, autoimmune disease of thetestis and ovary including autoimmune orchitis and oophoritis, primaryhypothyroidism, hypoparathyroidism, autoimmune endocrine diseasesincluding thyroiditis such as autoimmune thyroiditis, Hashimoto'sdisease, chronic thyroiditis (Hashimoto's thyroiditis), or subacutethyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism,Grave's disease, polyglandular syndromes such as autoimmunepolyglandular syndromes (or polyglandular endocrinopathy syndromes),paraneoplastic syndromes, including neurologic paraneoplastic syndromessuch as Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome,stiff-man or stiff-person syndrome, encephalomyelitis such as allergicencephalomyelitis or encephalomyelitis allergica and experimentalallergic encephalomyelitis (EAE), myasthenia gravis, cerebellardegeneration, neuromyotonia, opsoclonus or opsoclonus myoclonus syndrome(OMS), and sensory neuropathy, Sheehan's syndrome, autoimmune hepatitis,chronic hepatitis, lupoid hepatitis, giant cell hepatitis, chronicactive hepatitis or autoimmune chronic active hepatitis, lymphoidinterstitial pneumonitis, bronchiolitis obliterans (non-transplant) vsNSIP, Guillain-Barré syndrome, Berger's disease (IgA nephropathy),idiopathic IgA nephropathy, linear IgA dermatosis, primary biliarycirrhosis, pneumonocirrhosis, autoimmune enteropathy syndrome, Celiacdisease, Coeliac disease, celiac sprue (gluten enteropathy), refractorysprue, idiopathic sprue, cryoglobulinemia, amylotrophic lateralsclerosis (ALS; Lou Gehrig's disease), coronary artery disease,autoimmune inner ear disease (AIED) or autoimmune hearing loss,opsoclonus myoclonus syndrome (OMS), polychondritis such as refractoryor relapsed polychondritis, pulmonary alveolar proteinosis, amyloidosis,scleritis, a non-cancerous lymphocytosis, a primary lymphocytosis, whichincludes monoclonal B cell lymphocytosis (e.g., benign monoclonalgammopathy and monoclonal gammopathy of undetermined significance,MGUS), peripheral neuropathy, paraneoplastic syndrome, channelopathiessuch as epilepsy, migraine, arrhythmia, muscular disorders, deafness,blindness, periodic paralysis, and channelopathies of the CNS, autism,inflammatory myopathy, focal segmental glomerulosclerosis (FSGS),endocrine ophthalmopathy, uveoretinitis, chorioretinitis, autoimmunehepatological disorder, fibromyalgia, multiple endocrine failure,Schmidt's syndrome, adrenalitis, gastric atrophy, presenile dementia,demyelinating diseases such as autoimmune demyelinating diseases,diabetic nephropathy, Dressler's syndrome, alopecia arcata, CRESTsyndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility,sclerodactyl), and telangiectasia), male and female autoimmuneinfertility, mixed connective tissue disease, Chagas' disease, rheumaticfever, recurrent abortion, farmer's lung, erythema multiforme,post-cardiotomy syndrome, Cushing's syndrome, bird-fancier's lung,allergic granulomatous angiitis, benign lymphocytic angiitis, Alport'ssyndrome, alveolitis such as allergic alveolitis and fibrosingalveolitis, interstitial lung disease, transfusion reaction, leprosy,malaria, leishmaniasis, kypanosomiasis, schistosomiasis, ascariasis,aspergillosis, Sampter's syndrome, Caplan's syndrome, dengue,endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonaryfibrosis, interstitial lung fibrosis, idiopathic pulmonary fibrosis,cystic fibrosis, endophthalmitis, erythema elevatum et diutinum,erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome,Felty's syndrome, flariasis, cyclitis such as chronic cyclitis,heterochronic cyclitis, iridocyclitis, or Fuch's cyclitis,Henoch-Schonlein purpura, human immunodeficiency virus (HIV) infection,echovirus infection, cardiomyopathy, Alzheimer's disease, parvovirusinfection, rubella virus infection, post-vaccination syndromes,congenital rubella infection, Epstein-Barr virus infection, mumps,Evan's syndrome, autoimmune gonadal failure, Sydenham's chorea,post-streptococcal nephritis, thromboangitis ubiterans, thyrotoxicosis,tabes dorsalis, chorioiditis, giant cell polymyalgia, endocrineophthamopathy, chronic hypersensitivity pneumonitis,keratoconjunctivitis sicca, epidemic keratoconjunctivitis, idiopathicnephritic syndrome, minimal change nephropathy, benign familial andischemia-reperfusion injury, retinal autoimmunity, joint inflammation,bronchitis, chronic obstructive airway disease, silicosis, aphthae,aphthous stomatitis, arteriosclerotic disorders, aspermiogenese,autoimmune hemolysis, Boeck's disease, cryoglobulinemia, Dupuytren'scontracture, endophthalmia phacoanaphylactica, enteritis allergica,erythema nodosum leprosum, idiopathic facial paralysis, chronic fatiguesyndrome, febris rheumatica, Hamman-Rich's disease, sensoneural hearingloss, haemoglobinuria paroxysmatica, hypogonadism, ileitis regionalis,leucopenia, mononucleosis infectiosa, traverse myelitis, primaryidiopathic myxedema, nephrosis, ophthalmia symphatica, orchitisgranulomatosa, pancreatitis, polyradiculitis acuta, pyodermagangrenosum, Quervain's thyreoiditis, acquired spenic atrophy,infertility due to antispermatozoan antobodies, non-malignant thymoma,vitiligo, SCID and Epstein-Barr virus-associated diseases, acquiredimmune deficiency syndrome (AIDS), parasitic diseases such asLesihmania, toxic-shock syndrome, food poisoning, conditions involvinginfiltration of T cells, leukocyte-adhesion deficiency, immune responsesassociated with acute and delayed hypersensitivity mediated by cytokinesand T-lymphocytes, diseases involving leukocyte diapedesis, multipleorgan injury syndrome, antigen-antibody complex-mediated diseases,antiglomerular basement membrane disease, allergic neuritis, autoimmunepolyendocrinopathies, oophoritis, primary myxedema, autoimmune atrophicgastritis, sympathetic ophthalmia, rheumatic diseases, mixed connectivetissue disease, nephrotic syndrome, insulitis, polyendocrine failure,peripheral neuropathy, autoimmune polyglandular syndrome type I,adult-onset idiopathic hypoparathyroidism (AOIH), alopecia totalis,dilated cardiomyopathy, epidermolisis bullosa acquisita (EBA),hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosingcholangitis, purulent or nonpurulent sinusitis, acute or chronicsinusitis, ethmoid, frontal, maxillary, or sphenoid sinusitis, aneosinophil-related disorder such as eosinophilia, pulmonary infiltrationeosinophilia, eosinophilia-myalgia syndrome, Loffler's syndrome, chroniceosinophilic pneumonia, tropical pulmonary eosinophilia,bronchopneumonic aspergillosis, aspergilloma, or granulomas containingeosinophils, anaphylaxis, seronegative spondyloarthritides,polyendocrine autoimmune disease, sclerosing cholangitis, sclera,episclera, chronic mucocutaneous candidiasis, Bruton's syndrome,transient hypogammaglobulinemia of infancy, Wiskott-Aldrich syndrome,ataxia telangiectasia, autoimmune disorders associated with collagendisease, rheumatism, neurological disease, ischemic re-perfusiondisorder, reduction in blood pressure response, vascular dysfunction,antgiectasis, tissue injury, cardiovascular ischemia, hyperalgesia,cerebral ischemia, and disease accompanying vascularization, allergichypersensitivity disorders, glomerulonephritides, reperfusion injury,reperfusion injury of myocardial or other tissues, dermatoses with acuteinflammatory components, acute purulent meningitis or other centralnervous system inflammatory disorders, granulocytetransfusion-associated syndromes, cytokine-induced toxicity, acuteserious inflammation, chronic intractable inflammation, pyclitis,pncumonocirrhosis, diabetic retinopathy, diabetic large-artery disorder,endarterial hyperplasia, peptic ulcer, valvulitis, and endometriosis.

The term “detecting” is intended to include determining the presence orabsence of a substance or quantifying the amount of a substance. Theterm thus refers to the use of the materials, compositions, and methodsof the present invention for qualitative and quantitativedeterminations. In general, the particular technique used for detectionis not critical for practice of the invention.

For example, “detecting” according to the invention may include:observing the presence or absence of α5 gene product, a β1 gene product(e.g., mRNA molecules), or an α5 or α5β1 polypeptide; a change in thelevels of an α5 or α5β1 polypeptide or amount bound to a target; achange in biological function/activity of an α5 or α5β1 polypeptide. Insome embodiments, “detecting” may include detecting wild type α5 or α5β1levels (e.g., mRNA or polypeptide levels). Detecting may includequantifying a change (increase or decrease) of any value between 10% and90%, or of any value between 30% and 60%, or over 100%, when compared toa control. Detecting may include quantifying a change of any valuebetween 2-fold to 10-fold, inclusive, or more e.g., 100-fold.

“Label” when used herein refers to a detectable compound or compositionwhich is conjugated directly or indirectly to the antibody. The labelmay itself be detectable by itself (e.g., radioisotope labels orfluorescent labels) or, in the case of an enzymatic label, may catalyzechemical alteration of a substrate compound or composition which isdetectable.

III. Anti-α5β1 Antibodies

Antibodies that can bind human α5β1 and competitively inhibit thebinding of an anti-α5β1 antibody to human α5β1 are provided herein.Accordingly, one embodiment of the invention provides antibodiescomprising a variable light (VL) sequence set forth in any one of SEQ IDNOS: 1, 2, 3, or 4 and a variable heavy (VH) domain sequence set forthin any one of SEQ ID NOS: 5, 6, 7, 8, or 9. Human or chimeric forms ofthe antibodies of the deposited hybridomas are also contemplated.

According to one embodiment, the antibody binds a human α5β1 with a Kdbetween 500 nM and 1 pM. According to another embodiment, the antibodydoes not bind alphaVbeta3 or alphaVbeta5 or alphaVbeta1. According toanother embodiment, the antibody comprises a Fc sequence of a human IgG,e.g., human IgG1 or human IgG4. In another embodiment, a Fc sequence hasbeen altered or otherwise changed so that it that lacks antibodydependent cellular cytotoxicity (ADCC) effector function, often relatedto their binding to Fc receptors (FcRs). There are many examples ofchanges or mutations to Fc sequences that can alter effector function.For example, WO 00/42072 and Shields et al. J. Biol. Chem. 9(2):6591-6604 (2001) describe antibody variants with improved or diminishedbinding to FcRs. The contents of those publications are specificallyincorporated herein by reference. The antibody can be in the form of aFab, Fab′, a F(ab)′₂, single-chain Fv (scFv), an Fv fragment; a diabodyand a linear antibody. Also, the antibody can be a multi-specificantibody that binds to α5β1 and is an α5β1 antagonist, but also bindsone or more other targets and inhibits their function (e.g., VEGF). Theantibody can be conjugated to a therapeutic agent (e.g., cytotoxicagent, a radioisotope and a chemotherapeutic agent) or a label fordetecting α5β1 in patient samples or in vivo by imaging (e.g.,radioisotope, fluorescent dye and enzyme).

Nucleic acid molecules encoding the anti-α5β1 antibodies, expressionvectors comprising nucleic acid molecules encoding one or both variabledomains, and cells comprising the nucleic acid molecules are alsocontemplated. These antibodies can be used in the therapies describedherein and to detect α5β1 protein in patient samples (e.g., FACS,immunohistochemistry (IHC), ELISA assays) or in patients.

A. Monoclonal Antibodies

Monoclonal antibodies can be prepared, e.g., using hybridoma methods,such as those described by Kohler and Milstein, Nature, 256:495 (1975)or can be made by recombinant DNA methods (U.S. Pat. No. 4,816,567) orcan be produced by the methods described herein in the Examples below.In a hybridoma method, a hamster, mouse, or other appropriate hostanimal is typically immunized with an immunizing agent to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the immunizing agent. Alternatively, thelymphocytes can be immunized in vitro.

The immunizing agent will typically include a polypeptide or a fusionprotein of the protein of interest or a composition comprising theprotein. Generally, either peripheral blood lymphocytes (“PBLs”) areused if cells of human origin are desired, or spleen cells or lymph nodecells are used if non-human mammalian sources are desired. Thelymphocytes are then fused with an immortalized cell line using asuitable fusing agent, such as polyethylene glycol, to form a hybridomacell. Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (New York:Academic Press, 1986), pp. 59-103. Immortalized cell lines are usuallytransformed mammalian cells, particularly myeloma cells of rodent,bovine, and human origin. Usually, rat or mouse myeloma cell lines areemployed. The hybridoma cells can be cultured in a suitable culturemedium that preferably contains one or more substances that inhibit thegrowth or survival of the unfused, immortalized cells. For example, ifthe parental cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomastypically will include hypoxanthine, aminopterin, and thymidine (“HATmedium”), which substances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high-level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies. Kozbor, J. Immunol., 133:3001 (1984); Brodeur etal., MONOCLONAL ANTIBODY PRODUCTION TECHNIQUES AND APPLICATIONS (MarcelDekker, Inc.: New York, 1987) pp. 51-63.

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against thepolypeptide. The binding specificity of monoclonal antibodies producedby the hybridoma cells can be determined by immunoprecipitation or by anin vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA). Such techniques and assays are known inthe art. The binding affinity of the monoclonal antibody can, forexample, be determined by the Scatchard analysis of Munson and Pollard,Anal. Biochem., 107:220 (1980).

After the desired hybridoma cells are identified, the clones can besubcloned by limiting dilution procedures and grown by standard methods.Coding, supra. Suitable culture media for this purpose include, forexample, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.Alternatively, the hybridoma cells can be grown in vivo as ascites in amammal.

The monoclonal antibodies secreted by the subclones can be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

The monoclonal antibodies can also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells of theinvention serve as a preferred source of such DNA. Once isolated, theDNA can be placed into expression vectors, which are then transfectedinto host cells such as simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells. The DNA also can be modified, for example, bysubstituting the coding sequence for human heavy- and light-chainconstant domains in place of the homologous murine sequences (U.S. Pat.No. 4,816,567; Morrison et al., supra) or by covalently joining to theimmunoglobulin coding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptidecan be substituted for the constant domains of an antibody of theinvention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the invention to create achimeric bivalent antibody.

The antibodies can be monovalent antibodies. Methods for preparingmonovalent antibodies are known in the art. For example, one methodinvolves recombinant expression of immunoglobulin light chain andmodified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy-chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking.

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly Fabfragments, can be accomplished using, but not limited to, techniquesknown in the art.

B. Human and Humanized Antibodies

The antibodies can be humanized antibodies or human antibodies.Humanized forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains, or fragments thereof (such asFv, Fab, Fab′, F(ab′)₂, or other antigen-binding subsequences ofantibodies) that typically contain minimal sequence derived fromnon-human immunoglobulin. Humanized antibodies include humanimmunoglobulins (recipient antibody) in which residues from a CDR of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat, or rabbit having the desiredspecificity, affinity, and capacity. In some instances, Fv frameworkresidues of the human immunoglobulin are replaced by correspondingnon-human residues. Humanized antibodies can also comprise residues thatare found neither in the recipient antibody nor in the imported CDR orframework sequences. In general, the humanized antibody can comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin, and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody preferably also will comprise at least a portion ofan immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. Jones et al., Nature, 321: 522-525 (1986); Riechmann etal., Nature, 332: 323-329 (1988); Presta, Curr. Op. Struct. Biol.,2:593-596 (1992).

Some methods for humanizing non-human antibodies are described in theart and below in the Examples. Generally, a humanized antibody has oneor more amino acid residues introduced into it from a source that isnon-human. These non-human amino acid residues are often referred to as“import” residues, which are typically taken from an “import” variabledomain. According to one embodiment, humanization can be essentiallyperformed following the method of Winter and co-workers (Jones et al.,Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327(1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)), bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare antibodies (U.S. Pat. No. 4,816,567), wherein substantially lessthan an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

As an alternative to humanization, human antibodies can be generated.For example, it is now possible to produce transgenic animals (e.g.,mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (JH) genein chimeric and germ-line mutant mice results in complete inhibition ofendogenous antibody production. Transfer of the human germ-lineimmunoglobulin gene array into such germ-line mutant mice will result inthe production of human antibodies upon antigen challenge. See, e.g.,Jakobovits et al., PNAS USA, 90:2551 (1993); Jakobovits et al., Nature,362:255-258 (1993); Bruggemann et al., Year in Immunol., 7:33 (1993);U.S. Pat. Nos. 5,545,806, 5,569,825, 5,591,669; 5,545,807; and WO97/17852. Alternatively, human antibodies can be made by introducinghuman immunoglobulin loci into transgenic animals, e.g., mice in whichthe endogenous immunoglobulin genes have been partially or completelyinactivated. Upon challenge, human antibody production is observed thatclosely resembles that seen in humans in all respects, including generearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; and 5,661,016, and Marks et al.,Bio/Technology, 10: 779-783 (1992); Lonberg et al., Nature, 368: 856-859(1994); Morrison, Nature, 368: 812-813 (1994); Fishwild et al., NatureBiotechnology, 14: 845-851 (1996); Neuberger, Nature Biotechnology, 14:826 (1996); Lonberg and Huszar, Intern. Rev. Immunol., 13: 65-93 (1995).

Alternatively, phage display technology (McCafferty et al., Nature348:552-553 [1990]) can be used to produce human antibodies and antibodyfragments in vitro, from immunoglobulin variable (V) domain generepertoires from unimmunized donors. According to one embodiment of thistechnique, antibody V domain sequences are cloned in-frame into either amajor or minor coat protein gene of a filamentous bacteriophage, such asM13 or fd, and displayed as functional antibody fragments on the surfaceof the phage particle. Phage display can be performed in a variety offormats, e.g., as described below in the Examples section or as reviewedin, e.g., Johnson, Kevin S, and Chiswell, David J., Current Opinion inStructural Biology 3:564-571 (1993). Several sources of V-gene segmentscan be used for phage display. Clackson et al., Nature, 352:624-628(1991) isolated a diverse array of anti-oxazolone antibodies from asmall random combinatorial library of V genes derived from the spleensof immunized mice. A repertoire of V genes from unimmunized human donorscan be constructed and antibodies to a diverse array of antigens(including self-antigens) can be isolated essentially following thetechniques described by Marks et al., J. Mol. Biol. 222:581-597 (1991),or Griffith et al., EMBO J. 12:725-734 (1993). See, also, U.S. Pat. Nos.5,565,332 and 5,573,905.

As discussed above, human antibodies may also be generated by in vitroactivated B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275).

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries. Hoogenboom and Winter, J.Mol. Biol., 227: 381 (1991); Marks et al., J. Mol. Biol., 222: 581(1991). The techniques of Cole et al. and Boerner et al. are alsoavailable for the preparation of human monoclonal antibodies. Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1): 86-95 (1991).

C. Multi-Specific Antibodies

Multi-specific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for two or more differentantigens (e.g., bispecific antibodies have binding specificities for atleast two antigens). For example, one of the binding specificities canbe for the α5β1 protein, the other one can be for any other antigen.According to one preferred embodiment, the other antigen is acell-surface protein or receptor or receptor subunit. For example, thecell-surface protein can be a natural killer (NK) cell receptor. Thus,according to one embodiment, a bispecific antibody of this invention canbind both α5β1 and VEGF.

Suitable methods for making bispecific antibodies are well known in theart. For example, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities. Milsteinand Cuello, Nature, 305: 537-539 (1983). Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of ten different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule is usually accomplished by affinitychromatography steps. Similar procedures are disclosed in WO 93/08829and in Traunecker et al., EMBO., 10: 3655-3659 (1991).

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant-domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies, see, for example,Suresh et al., Methods in Enzymology, 121: 210 (1986).

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., PNAS USA,90:6444-6448 (1993) has provided an alternative mechanism for makingbispecific antibody fragments. The fragments comprise a VH connected toa VL by a linker which is too short to allow pairing between the twodomains on the same chain. Accordingly, the VH and VL domains of onefragment are forced to pair with the complementary VL and VH domains ofanother fragment, thereby forming two antigen-binding sites. Anotherstrategy for making bispecific antibody fragments by the use ofsingle-chain Fv (sFv) dimers has also been reported. See Gruber et al.,J. Immunol., 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60(1991).

D. Heteroconjugate Antibodies Heteroconjugate antibodies are composed oftwo covalently joined antibodies. Such antibodies have, for example,been proposed to target immune-system cells to unwanted cells (U.S. Pat.No. 4,676,980), and for treatment of HIV infection. WO 91/00360; WO92/200373; EP 03089. It is contemplated that the antibodies can beprepared in vitro using known methods in synthetic protein chemistry,including those involving crosslinking agents. For example, immunotoxinscan be constructed using a disulfide-exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

E. Effector Function Engineering

It can be desirable to modify the antibody of the invention with respectto effector function, so as to enhance, e.g., the effectiveness of theantibody in treating cancer. For example, cysteine residue(s) can beintroduced into the Fc region, thereby allowing interchain disulfidebond formation in this region. The homodimeric antibody thus generatedcan have improved internalization capability and/or increasedcomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC). See, Caron et al., J. Exp. Med., 176: 1191-1195(1992) and Shopes, J. Immunol., 148: 2918-2922 (1992). Homodimericantibodies with enhanced anti-tumor activity can also be prepared usingheterobifunctional cross-linkers as described in Wolff et al., CancerResearch, 53: 2560-2565 (1993). Alternatively, an antibody can beengineered that has dual Fc regions and can thereby have enhancedcomplement lysis and ADCC capabilities. See, Stevenson et al.,Anti-Cancer Drug Design, 3: 219-230 (1989).

Mutations or alterations in the Fc region sequences can be made toimprove FcR binding (e.g., FcgammaR, FcRn). According to one embodiment,an antibody of this invention has at least one altered effector functionselected from the group consisting of ADCC, CDC, and improved FcRnbinding compared to a native IgG or a parent antibody. Examples ofseveral useful specific mutations are described in, e.g., Shields, R Let al. (2001) JBC 276(6)6591-6604; Presta, L. G., (2002) BiochemicalSociety Transactions 30(4):487-490; and WO 00/42072.

According to one embodiment, the Fc receptor mutation is a substitutionat least one position selected from the group consisting of: 238, 239,246, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272,276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298,301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330,331, 332, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388,389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439 of the Fcregion, wherein the numbering of the residues in the Fc region isaccording to the EU numbering system. In some embodiments, the Fcreceptor mutation is a D265A substitution. In some embodiments, the Fcreceptor mutation is a N297A substitution. Additional suitable mutationsare set forth in U.S. Pat. No. 7,332,581.

F. Immunoconjugates

The invention also pertains to immunoconjugates comprising an antibodyconjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin(e.g., an enzymatically active toxin of bacterial, fungal, plant, oranimal origin, or fragments thereof), or a radioactive isotope (i.e., aradioconjugate).

Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Enzymatically active toxinsand fragments thereof that can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. Avariety of radionuclides are available for the production ofradioconjugated antibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y,and ¹⁸⁶Re.

Conjugates of the antibody and cytotoxic agent are made using a varietyof bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCl), active esters (such as disuccinimidyl suberate),aldehydes (such as glutaraldehyde), bis-azido compounds (such asbis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science, 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See, WO94/11026.

In another embodiment, the antibody can be conjugated to a “receptor”(such as streptavidin) for utilization in tumor pretargeting wherein theantibody-receptor conjugate is administered to the patient, followed byremoval of unbound conjugate from the circulation using a clearing agentand then administration of a “ligand” (e.g., avidin) that is conjugatedto a cytotoxic agent (e.g., a radionucleotide).

G. Immunoliposomes

The antibodies disclosed herein can also be formulated asimmunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., PNAS USA,82: 3688 (1985); Hwang et al., PNAS USA, 77: 4030 (1980); and U.S. Pat.Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation timeare disclosed in U.S. Pat. No. 5,013,556.

Particularly useful liposomes can be generated by the reverse-phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol, and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem., 257: 286-288 (1982) via a disulfide-interchange reaction. Ananti-neoplastic agent, a growth inhibitory agent, or a chemotherapeuticagent (such as Doxorubicin) is optionally also contained within theliposome. See, Gabizon et al., J. National Cancer Inst., 81(19): 1484(1989).

IV. Methods of Treatment Using Anti-α5β1 Antibodies

The anti-α5β1 antibodies of the invention can be administered tosubjects (e.g., mammals such as humans) to treat diseases and disordersinvolving abnormal angiogenesis and/or vascular permeability or leakage,including, for example, cancer, ocular diseases, and immune disorders(e.g., autoimmune disorders). Administration can be by any suitableroute including, e.g., intravenous, intramuscular, or subcutaneous.

In some embodiments, the anti-α5β1 antibodies of the invention areadministered in combination with a second, third, or fourth agent(including, e.g., an anti-neoplastic agent, a growth inhibitory agent, acytotoxic agent, or a chemotherapeutic agent) to treat the diseases ordisorders involving abnormal angiogenesis and/or vascular permeabilityor leakage. In some embodiments, the anti-α5β1 antibodies are conjugatedto the additional agent.

In some embodiments, the anti-α5β1 antibodies are administered incombination with a VEGF antagonist. The anti-α5β1 antibody andadditional agent (e.g., a VEGF antagonist) can be administeredconcurrently or sequentially. Alternatively, the subject can be treatedwith the VEGF antagonist and subsequently administered the α5β1antagonist, e.g., treating with the VEGF antagonist until the subject isunresponsive to VEGF antagonist treatment and then treating the subjectis treated with an α5β1 antagonist. According to one embodiment, thesubject is treated with the VEGF antagonist when the cancer isnon-invasive and then treated with the α5β1 antagonist when the canceris invasive. Some patients who experience elevated α5β1 levels naturallyor in response to VEGF antagonist therapy, compared to non-diseasedpatients or control, can be especially responsive to this combinationtreatment. Combinations further comprising a therapeutic agent (e.g., ananti-neoplastic agent, a chemotherapeutic agent, a growth inhibitoryagent and a cytotoxic agent) are contemplated. For example, patients whoare to be treated with chemotherapy (e.g., irinotecan) and α5β1antagonists, or who have been treated with chemotherapy and α5β1antagonists, can benefit from VEGF antagonist therapy. Alternatively,patients who have been treated with chemotherapy and VEGF antagonistscan benefit from α5β1 antagonist therapy. In one preferred embodiment,the anti-VEGF antibody is the Avastin® antibody. In another preferredembodiment, the anti-α5β1 antibody is an anti-α5β1 antibody describedherein. Kits comprising a VEGF antagonist, an α5β1 antagonist and,optionally, a chemotherapeutic agent are contemplated.

Cancer treatments can be evaluated by, e.g., but not limited to, tumorregression, tumor weight or size shrinkage, time to progression,duration of survival, progression free survival, overall response rate,duration of response, quality of life, protein expression and/oractivity. Because the anti-angiogenic agents described herein target thetumor vasculature and not necessarily the neoplastic cells themselves,they represent a unique class of anticancer drugs, and therefore canrequire unique measures and definitions of clinical responses to drugs.For example, tumor shrinkage of greater than 50% in a 2-dimensionalanalysis is the standard cut-off for declaring a response. However, theα5β1 antagonists and VEGF antagonists of the invention may causeinhibition of metastatic spread without shrinkage of the primary tumor,or may simply exert a tumouristatic effect. Accordingly, approaches todetermining efficacy of the therapy can be employed, including forexample, measurement of plasma or urinary markers of angiogenesis andmeasurement of response through radiological imaging.

Depending on the indication to be treated and factors relevant to thedosing that a physician of skill in the field would be familiar with,the antibodies of the invention will be administered at a dosage that isefficacious for the treatment of that indication while minimizingtoxicity and side effects. For the treatment of a cancer, an autoimmunedisease or an immunodeficiency disease, the therapeutically effectivedosage can be, e.g., in the range of 50 mg/dose to 2.5 g/m2. In oneembodiment, the dosage administered is about 250 mg/m2 to about 400mg/m2 or 500 mg/m2. In another embodiment, the dosage is about 250-375mg/m2. In yet another embodiment, the dosage range is 275-375 mg/m2.

Evaluation of treatments for age-related macular degeneration (AMD)includes, but it is not limited to, a decrease in the rate of furthervision loss or the prevention of further vision loss. For AMD therapy,efficacy in vivo can, for example, be measured by one or more of thefollowing: assessing the mean change in the best corrected visual acuity(BCVA) from baseline to a desired time, assessing the proportion ofsubjects who lose fewer than 15 letters in visual acuity at a desiredtime compared with baseline, assessing the proportion of subjects whogain greater than or equal to 15 letters in visual acuity at a desiredtime compared with baseline, assessing the proportion of subjects with avisual-acuity Snellen equivalent of 20/2000 or worse at desired time,assessing the NEI Visual Functioning Questionnaire, assessing the sizeof CNV and amount of leakage of CNV at a desired time, as assessed byfluorescein angiography, and the like.

V. Pharmaceutical Formulations

The anti-α5β1 antibodies can be formulated with suitable carriers orexcipients so that they are suitable for administration. Suitableformulations of the antibodies are obtained by mixing an antibody havingthe desired degree of purity with optional pharmaceutically acceptablecarriers, excipients or stabilizers (Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980)), in the form of lyophilizedformulations or aqueous solutions. Acceptable carriers, excipients, orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as olyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG). Exemplary antibody formulations are describedin WO98/56418, expressly incorporated herein by reference. Lyophilizedformulations adapted for subcutaneous administration are described inWO97/04801. Such lyophilized formulations may be reconstituted with asuitable diluent to a high protein concentration and the reconstitutedformulation may be administered subcutaneously to the mammal to betreated herein.

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.For example, it may be desirable to further provide an anti-neoplasticagent, a growth inhibitory agent, a cytotoxic agent, or achemotherapeutic agent. Such molecules are suitably present incombination in amounts that are effective for the purpose intended. Theeffective amount of such other agents depends on the amount of antibodypresent in the formulation, the type of disease or disorder ortreatment, and other factors discussed above. These are generally usedin the same dosages and with administration routes as described hereinor about from 1 to 99% of the heretofore employed dosages.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semi-permeable matrices of solidhydrophobic polymers containing the antagonist, which matrices are inthe form of shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid andethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.

Lipofectins or liposomes can be used to deliver the polypeptides andantibodies or compositions of this invention into cells. Where antibodyfragments are used, the smallest inhibitory fragment that specificallybinds to the binding domain of the target protein is preferred. Forexample, based upon the variable-region sequences of an antibody,peptide molecules can be designed that retain the ability to bind thetarget protein sequence. Such peptides can be synthesized chemicallyand/or produced by recombinant DNA technology. See, e.g., Marasco etal., PNAS USA, 90: 7889-7893 (1993).

The active ingredients can also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles, andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's PHARMACEUTICAL SCIENCES, supra.

Sustained-release preparations can be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated antibodies remainin the body for a long time, they can denature or aggregate as a resultof exposure to moisture at 37° C., resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS—S bond formation through thio-disulfide interchange, stabilization canbe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

VI. Methods of Diagnosis and Imaging Using Anti-α5β1 Antibodies

Labeled anti-α5β1 antibodies, and derivatives and analogs thereof, whichspecifically bind to an α5β1 polypeptide can be used for diagnosticpurposes to detect, diagnose, or monitor diseases and/or disordersassociated with the expression, aberrant expression and/or activity ofα5β1. For example, the anti-α5β1 antibodies of the invention can be usedin in situ, in vivo, ex vivo, and in vitro diagnostic assays or imagingassays.

Methods for detecting expression of an α5β1 polypeptide, comprising (a)assaying the expression of the polypeptide in cells (e.g., tissue) orbody fluid of an individual using one or more antibodies of thisinvention and (b) comparing the level of gene expression with a standardgene expression level, whereby an increase or decrease in the assayedgene expression level compared to the standard expression level isindicative of aberrant expression.

Additional embodiments of the invention include methods of diagnosing adisease or disorder associated with expression or aberrant expression ofα5β1 in an animal (e.g., a mammal such as a human). The methods comprisedetecting α5β1 molecules in the mammal. In one embodiment, afteradministering a VEGF antagonist, diagnosis comprises: (a) administeringan effective amount of a labeled anti-α5β1 antibody to a mammal (b)waiting for a time interval following the administering for permittingthe labeled α5β1 antibody to preferentially concentrate at sites in thesubject where the α5β1 molecule is expressed (and for unbound labeledmolecule to be cleared to background level); (c) determining backgroundlevel; and (d) detecting the labeled molecule in the subject, such thatdetection of labeled molecule above the background level indicates thatthe subject has a particular disease or disorder associated withexpression or aberrant expression of α5β1. Background level can bedetermined by various methods including, comparing the amount of labeledmolecule detected to a standard value previously determined for aparticular system.

Anti-α5β1 antibodies of the invention can be used to assay proteinlevels in a biological sample using classical immunohistological methodsknown to those of skill in the art (e.g., see Jalkanen, et al., J. Cell.Biol. 101:976-985 (1985); Jalkanen, et al., J. Cell. Biol. 105:3087-3096(1987)). Other antibody-based methods useful for detecting protein geneexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assaylabels are known in the art and include enzyme labels, such as, glucoseoxidase; radioisotopes, such as iodine (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I), carbon(¹⁴C) sulfur (³⁵S), tritium (³H), indium (^(115m)In, ^(113m)In, ¹¹²In,¹¹¹In), and technetium (⁹⁹Tc, ^(99m)Tc), thallium (²⁰¹Ti), gallium(⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe),fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y,⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru; luminol; and fluorescent labels,such as fluorescein and rhodamine, and biotin.

Techniques known in the art may be applied to labeled antibodies of theinvention. Such techniques include, but are not limited to, the use ofbifunctional conjugating agents (see e.g., U.S. Pat. Nos. 5,756,065;5,714,631; 5,696,239; 5,652,361; 5,505,931; 5,489,425; 5,435,990;5,428,139; 5,342,604; 5,274,119; 4,994,560; and 5,808,003).

According to one specific embodiment, and polypeptide expression oroverexpression is determined in a diagnostic or prognostic assay afteradministration of a VEGF antagonist therapeutic agent by evaluatinglevels of α5β1 present on the surface of a cell (e.g., via animmunohistochemistry assay using anti-α5β1 antibodies). Alternatively,or additionally, one can measure levels of α5β1 polypeptide-encodingnucleic acid or mRNA in the cell, e.g., via fluorescent in situhybridization using a nucleic acid based probe corresponding to anα5β1-encoding nucleic acid or the complement thereof; (FISH; seeWO98/45479 published October, 1998), Southern blotting, Northernblotting, or polymerase chain reaction (PCR) techniques, such as realtime quantitative PCR (RT-PCR). One can also study α5β1 overexpressionby measuring shed antigen in a biological fluid such as serum, e.g.,using antibody-based assays (see also, e.g., U.S. Pat. No. 4,933,294issued Jun. 12, 1990; WO91/05264 published Apr. 18, 1991; U.S. Pat. No.5,401,638 issued Mar. 28, 1995; and Sias et al., J. Immunol. Methods132:73-80 (1990)). Aside from the above assays, various in vivo and exvivo assays are available to the skilled practitioner. For example, onecan expose cells within the body of the mammal to an antibody which isoptionally labeled with a detectable label, e.g., a radioactive isotope,and binding of the antibody to the can be evaluated, e.g., by externalscanning for radioactivity or by analyzing a sample (e.g., a biopsy orother biological sample) taken from a mammal previously exposed to theantibody.

VII. Articles of Manufacture and Kits

Another embodiment of the invention is an article of manufacturecontaining materials useful for the treatment of cancer (e.g. tumors),ocular disease (e.g., wet AMD) or autoimmune diseases and relatedconditions. The article of manufacture can comprise a container and alabel or package insert on or associated with the container. Suitablecontainers include, for example, bottles, vials, syringes, etc. Thecontainers may be formed from a variety of materials such as glass orplastic. Generally, the container holds a composition which is effectivefor treating the condition and may have a sterile access port (forexample the container may be an intravenous solution bag or a vialhaving a stopper pierceable by a hypodermic injection needle). At leastone active agent in the composition is a VEGF antagonist or an α5β1antagonist or an VEGF agonist or an α5β1 agonist of the invention. Thelabel or package insert indicates that the composition is used fortreating the particular condition. The label or package insert willfurther comprise instructions for administering the antibody compositionto the patient. Articles of manufacture and kits comprisingcombinatorial therapies described herein are also contemplated.

Package insert refers to instructions customarily included in commercialpackages of therapeutic products, that contain information about theindications, usage, dosage, administration, contraindications and/orwarnings concerning the use of such therapeutic products. In oneembodiment, the package insert indicates that the composition is usedfor treating non-Hodgkins' lymphoma.

Additionally, the article of manufacture may further comprise a secondcontainer comprising a pharmaceutically-acceptable buffer, such asbacteriostatic water for injection (BWFI), phosphate-buffered saline,Ringer's solution and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, and syringes.

Kits are also provided that are useful for various purposes, e.g., forisolation or detection of α5β1 and/or VEGF in patients, optionally incombination with the articles of manufacture. For isolation andpurification of α5β1, the kit can contain an anti-α5β1 antibody coupledto beads (e.g., sepharose beads). Kits can be provided which contain theantibodies for detection and quantitation of α5β1 and/or VEGF in vitro,e.g. in an ELISA or a Western blot. As with the article of manufacture,the kit comprises a container and a label or package insert on orassociated with the container. For example, the container holds acomposition comprising at least one anti-α5β1 antibody of the invention.Additional containers may be included that contain, e.g., diluents andbuffers, control antibodies. The label or package insert may provide adescription of the composition as well as instructions for the intendedin vitro or diagnostic use.

Commercially available reagents referred to in the Examples were usedaccording to manufacturer's instructions unless otherwise indicated. Thesource of those cells identified in the following Examples, andthroughout the specification, by ATCC accession numbers is the AmericanType Culture Collection, Manassas, Va. Unless otherwise noted, thepresent invention uses standard procedures of recombinant DNAtechnology, such as those described hereinabove and in the followingtextbooks: Sambrook et al., supra; Ausubel et al., CURRENT PROTOCOLS INMOLECULAR BIOLOGY (Green Publishing Associates and Wiley Interscience,N.Y., 1989); Innis et al., PCR PROTOCOLS: A GUIDE TO METHODS ANDAPPLICATIONS (Academic Press, Inc.: N.Y., 1990); Harlow et al.,ANTIBODIES: A LABORATORY MANUAL (Cold Spring Harbor Press: Cold SpringHarbor, 1988); Gait, OLIGONUCLEOTIDE SYNTHESIS (IRL Press: Oxford,1984); Freshney, ANIMAL CELL CULTURE, 1987; Coligan et al., CURRENTPROTOCOLS IN IMMUNOLOGY, 1991.

All publications (including patents and patent applications) citedherein are hereby incorporated in their entirety by reference, includingspecifically, U.S. Provisional Application No. 60/784,704, filed Mar.21, 2006, U.S. Provisional Application No. 60/785,330, filed Mar. 22,2006; and U.S. Provisional Application No. 60/871,743, filed Dec. 22,2006.

The following DNA sequences were deposited under the terms of theBudapest Treaty with the American Type Culture Collection (ATCC), 10801University Blvd., Manassas, Va. 20110-2209, USA as described below:

Material Deposit No. Deposit Date Alpha5/beta1 7H5.4.2.8 PTA-7421 Mar.7, 2006

The deposits herein were made under the provisions of the BudapestTreaty on the International Recognition of the Deposit of Microorganismsfor the Purpose of Patent Procedure and the Regulations thereunder(Budapest Treaty). This assures maintenance of a viable culture of thedeposits for 30 years from the date of deposit. The deposits will bemade available by ATCC under the terms of the Budapest Treaty, andsubject to an agreement between Genentech, Inc. and ATCC, which assurespermanent and unrestricted availability of the progeny of the culture

of the deposits to the public upon issuance of the pertinent U.S. patentor upon laying open to the public of any U.S. or foreign patentapplication, whichever comes first, and assures availability of theprogeny to one determined by the U.S. Commissioner of Patents andTrademarks to be entitled thereto according to 35 U.S.C. 122 and theCommissioner's rules pursuant to thereto (including 37 C.F.R. 1.14 withparticular reference to 886 OG 638).

The assignee of the present application has agreed that if a culture ofthe materials on deposits should die or be lost or destroyed whencultivated under suitable conditions, the materials will be promptlyreplaced on notification with another of the same. Availability of thedeposited material is not to be construed as a license to practice theinvention in contravention of the rights granted under the authority ofany government in accordance with its patent laws.

Commercially available reagents referred to in the Examples were usedaccording to manufacturer's instructions unless otherwise indicated. Thesource of those cells identified in the following Examples, andthroughout the specification, by ATCC accession numbers is the AmericanType Culture Collection, Manassas, Va. Unless otherwise noted, thepresent invention uses standard procedures of recombinant DNAtechnology, such as those described hereinabove and in the followingtextbooks: Sambrook et al., supra; Ausubel et al., Current Protocols inMolecular Biology (Green Publishing Associates and Wiley Interscience,N.Y., 1989); Innis et al., PCR Protocols: A Guide to Methods andApplications (Academic Press, Inc.: N.Y., 1990); Harlow et al.,Antibodies: A Laboratory Manual (Cold Spring Harbor Press: Cold SpringHarbor, 1988); Gait, Oligonucleotide Synthesis (IRL Press: Oxford,1984); Freshney, Animal Cell Culture, 1987; Coligan et al., CurrentProtocols in Immunology, 1991.

The foregoing written description is considered to be sufficient toenable one skilled in the art to practice the invention. The followingExamples are offered for illustrative purposes only, and are notintended to limit the scope of the present invention in any way. Indeed,various modifications of the invention in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and fall within the scope of the appendedclaims.

Examples (1) Direct CDR Grafts onto the Acceptor Human ConsensusFramework

The phagemid used for this work is a monovalent Fab-g3 display vectoressentially as described in Lee et al., J. Mol. Biol. 340:1073-93(2004). Hypervariable sequences from the mouse monoclonal antibody 7H5sequences were grafted onto the human consensus kappa I (huKI) and humansubgroup III consensus VH (huIII) domains. To make the CDR graft, theacceptor VH framework, which differs from the human subgroup IIIconsensus VH domain at 3 positions: R71A, N73T, and L78A (Carter et al.,Proc. Natl. Acad. Sci. USA 89:4285 (1992)) was used.

(2) Modification of CDRs Asparagines of Humanized 7H5.v1

Substitutions with other resides at various positions was achieved byKunkel mutagenesis using a separate oligonucleotide for each position.Phage competition ELISA was utilized for evaluating all of the variants,monovalently displayed as a Fab on the phage, binding affinities (IC50)against human integrin α5β1 in FIG. 4. The relative fold of losing orimproving binding affinity as compared to parental humanized 7H5.v1 wassummarized in FIG. 5. The IC50 values for binding affinity weredetermined by phage competition ELISA in FIG. 6. Two variants, h7H5.v2and h7H5.v3 as indicated were selected to include glycine substitutionat position 65 of CDR-H2, cloned into hIgG1 vector, and evaluatedbinding affinities using BIAcore instrument. In FIG. 7, the BIAcorebinding analysis indicated humanized 7H5.v2 indeed slightly improvedbinding affinity to a level of sub-nanomolar.

(3) Framework Modifications of Humanized 7H5.v2

The following positions were mutated: light chain position 46 (T46L),heavy chain positions 48 (148V), 49 (G49S), 66 (K66R), 67 (A67F), 69(L69I) and 78 (A78L) in h7H5.v2. In addition, the heavy chain position30 (T305) was mutated as well.

Mutation at each position was generated by Kunkel mutagenesis using aseparate oligonucleotide. Phage competition ELISA was utilized forevaluating all of the variants. Monovalent Fab was displayed on thephage. Binding affinities (IC50) against human integrin α5β1 wasdetermined. FIG. 8 shows that most of the mutations still retained thesame level of binding affinities as compared to the parental clone,except for the two variants with changes at heavy chain positions 49(G49S) and 78 (A78L).

Therefore, humanized 7H5.v4 and v5 were generated by including all ofthe following mutations, light chain position 46 (T46L), heavy chainpositions 30 (T305), 48 (148V), 66 (K66R), 67 (A67F) and 69 (L69I), andonly differed at heavy chain position 49. The binding affinity for bothvariants was determined using BIAcore instrument. FIG. 9 shows thath7H5.v4 retained the same level of binding affinity as its parentalclone humanized 7H5.v2.

(4) Phage Competition ELISA

MAXISORP™ microtiter plates were coated with recombinant human integrinα5β1 (R&D) at 5 μg/ml in PBS overnight and then blocked with PBST buffer(0.5% BSA and 0.05% Tween 20 in PBS) for an hour at room temperature.Phage from culture supernatants were incubated with serially dilutedhuman integrin α5β1 in PBST buffer in a tissue-culture microtiter platefor an hour, after which 80 μl of the mixture was transferred to thetarget-coated wells for 15 minutes to capture unbound phage. The platewas washed with PBT buffer (0.05% Tween 20 in PBS), and HRP-conjugatedanti-M13 (Amersham Pharmacia Biotech) was added (1:5000 in PBST buffer)for 40 minutes. The plate was washed with PBT buffer and developed byadding tetramethylbenzidine substrate (Kirkegaard and PerryLaboratories, Gaithersburg, Md.). The absorbance at 450 nm was plottedas a function of target concentration in solution to determine phageIC₅₀. This was used as an affinity estimate for the Fab clone displayedon the surface of the phage.

(5) IgG Production and Antibody Affinity Determinations by BIAcoreExperiment

Clones of interest (Chimeric 7H5, humanized 7H5.v1, v2, v3, v4, and v5)were reformatted into a human IgG1 pRK vector (Carter et al., PNAS USA,89: 4285-4289 (1992)), transiently expressed in CHO cells, and purifiedwith Protein A affinity chromatography.

Affinity determinations were performed by surface-plasmon resonanceusing a BIACORE™-3000 instrument. Immobilization was achieved by randomcoupling through amino groups using a protocol provided by themanufacturer. Two different formats were operated to study bindingkinetics for antibody against human integrin α5β1. In FIGS. 3A, 7, and9A, antibodies were immobilized on a CM5 sensor chip around 450 responseunits (RU), and 2-fold serial diluted concentrations of human integrinα5β1 (300 nM to 0.29 nM) in PBT buffer (0.05% Tween 20 in PBS) wereinjected with a flow rate of 30 μl/min at 25° C. In FIGS. 3B and 9B,human integrin α5β1 was immobilized on a CM5 sensor chip around 800 RU,and 2-fold serial diluted concentrations of antibodies (200 nM to 0.2nM) in PBT buffer (0.05% Tween 20 in PBS) were injected with a flow rateof 30 μl/min at 25° C. After each injection, the chip was regeneratedusing 10 mM Glycine-HCl buffer at pH 1.7. Binding response was correctedby subtracting the RU from a blank flow cell. Association rates (k_(on))and dissociation rates (k_(off)) were calculated using a simpleone-to-one Langmuir binding model (BIAcore Evaluation Software version3.2). The equilibrium dissociation constant (K_(d)) was calculated asthe ratio k_(off)/k_(on).

(6) Skin Wound Healing Assays

New Zealand White rabbits were used in studies to demonstrate theefficacy of humanized 7H5 antibodies. Using aseptic technique, acircular 8 mm biopsy punch was used to produce wounds to the depth ofthe ear cartilageand the underlying perichondrium is removed with aperiosteal elevator and fine scissors. Gross appearance of each woundwas monitored daily until the end of the study. Wound gap measurementswere taken on days 0, 7, 14, 21, and 28. All animals were euthanized onday 28. Two sets of skin wound healing experiments were conducted.

The first used the following study groups, with 5 animals per group andtwo wounds per animal: (1) negative control IgG (200 μg/30 μl/wound perday); (2) h7H5.v2 (100 μg/30 μl/wound per day); (3) anti-VEGF antibody(100 μg/30 μl/wound per day); and (4) h7H5.v2 (100 μg/15 μl/wound perday) in combination with anti-VEGF antibody (100 μg/15 μl/wound perday). The antibodies were applied topically. FIG. 10 depicts resultsdemonstrating that administration of (1) h7H5.V2 alone or anti-VEGFalone inhibits wound healing; and (2) the combined administration ofh7H5.v2 and anti-VEGF antibody enhances the effects of anti-VEGFantibody alone on wound healing.

The second set of experiments used the following study groups, with 5animals per group and two wounds per animal: (1) negative control IgG(200 μg/30 μl/wound per day); (2) h7H5.v4 (100 μg/30 μl/wound per day);(3) anti-VEGF antibody (100 μg/30 μl/wound per day); and (4) h7H5.v4(100 μg/15 μl/wound per day) in combination with anti-VEGF antibody (100μg/15 μl/wound per day). The antibodies were applied topically. FIG. 11depicts results demonstrating that administration of (1) h7H5.V4 oranti-VEGF alone inhibits wound healing; and (2) the combinedadministration of h7H5.v4 and anti-VEGF antibody enhances the effects ofanti-VEGF antibody alone on wound healing.

All publications (including, e.g., patents, published patentapplications, and Genbank Accession Nos.) cited herein are herebyincorporated by reference in their entirety for all purposes as if eachreference were specifically and individually incorporated by reference.

1-28. (canceled) 29: A method for treating cancer in a subjectcomprising administering a VEGF antagonist and an anti-α5β1 antibody,wherein the anti-α5β1 antibody comprises a light chain variable domainsequence comprising (1) an LHVR1 comprising the amino acid sequenceKASQ-N/S-VGSDVA (SEQ ID NO:10), (2) an LHVR2 comprising the amino acidsequence STSYRYS (SEQ ID NO:11) and (3) an LHVR3 comprising the aminoacid sequence QQY-N/S-SYPFT (SEQ ID NO:12) and a heavy chain variabledomain sequence comprising (1) an HHVR1 comprising the amino acidsequence GYTF-T/S-DYYLY (SEQ ID NO:13), (2) an HHVR2 comprising theamino acid sequence GISPS-N/S-GGTTF-N/A-D-N/A-FE-N/G (SEQ ID NO:14) and(3) an HHVR3 comprising the amino acid sequence DAYGDWYFDV (SEQ IDNO:15). 30-32. (canceled) 33: The method of claim 29, wherein thesubject is further administered a therapeutic agent selected from thegroup consisting of an anti-neoplastic agent, a chemotherapeutic agent,a growth inhibitory agent and a cytotoxic agent. 34: The method of claim29, wherein the VEGF antagonist can be competitively inhibited frombinding to human VEGF by the Avastin® antibody. 35: The method accordingto claim 34, wherein the VEGF antagonist is the Avastin® antibody. 36.(canceled) 37: The method of claim 29, wherein the anti-α5β1 antibodycomprises a light chain variable domain having the sequence set forth inany one of SEQ ID NOS:1, 2, 3, or 4 or variant there of and a heavychain variable domain having the sequence set forth in any one of SEQ IDNOS:5, 6, 7, 8, or 9 or variant thereof. 38: The method of claim 37,wherein the light chain variable domain has the sequence set forth inSEQ ID NO: 3 and a heavy chain variable domain having the sequence setforth in SEQ ID NO:8. 39: The method of claim 37, wherein the variantsequence of the heavy chain variable domain comprises an amino acidsubstitution at a residue selected from the group consisting of 30, 48,49, 54, 60, 62, 65, 66, 67 and
 69. 40: The method of claim 37, whereinthe light chain variable domain comprises an amino acid substitution ata residue selected from the group consisting of 28, 46 and
 92. 41: Themethod of claim 39, wherein the amino acid substitution is selected fromthe group consisting of T30S, I48V, G49S, N54S, N60A, N62A, N62S, N65G,K66R, A67F and L69I. 42: The method of claim 40, wherein the light chainvariable domain comprises an amino acid substitution selected from thegroup consisting of N28S, T46L and N92S. 43: The method of claim 29,wherein the anti-α5β1 antibody does not bind alphaVbeta3 or alphaVbeta5or alphaVbeta1. 44: The method of claim 29, wherein the anti-α5β1antibody comprises an Fc sequence of a human IgG. 45: The method ofclaim 44, wherein the human IgG is hIgG1 or hIgG4. 46: The method ofclaim 44, wherein the anti-α5β1 antibody comprises a Fc sequence thatlacks antibody dependent cellular cytotoxicity (ADCC) effector function.47: The method of claim 46, wherein the Fc sequence comprises a D265Asubstitution. 48: The method of claim 29, wherein the anti-α5β1 antibodyis selected from the group consisting of a Fab, Fab′, a F(ab)′₂,single-chain Fv (scFv), an Fv fragment, a diabody and a linear antibody.49: The method of claim 29, wherein the anti-α5β1 antibody is amulti-specific antibody. 50: The method of claim 29, wherein theanti-α5β1 antibody is conjugated to a therapeutic agent. 51: The methodof claim 50, wherein the therapeutic agent is selected from the groupconsisting of a cytotoxic agent, a radioisotope and a chemotherapeuticagent. 52: The method of claim 29, wherein the anti-α5β1 antibody isconjugated to a label. 53: The method of claim 52, wherein the label isselected from the group consisting of a radioisotope, fluorescent dyeand enzyme.